Method of improving the production of biomass or a desired product from a cell

ABSTRACT

The production of biomass or a desired product from a cell can be improved by inducing conversion of ATP to ADP without primary effects on other cellular metabolites or functions which is achieved by expressing an uncoupled ATPase activity in said cell and incubating the cell with a suitable substrate to produce said biomass or product. This is conveniently done by expressing in said cell the soluble part (F 1 ) of the membrane bound (F 0 F 1  type) H + -ATPase or a portion of F 1  exhibiting ATPase activity. The organism from which the F 1  ATPase or portions thereof is derived, or in which the F 1  ATPase or portions thereof is expressed, may be selected from prokaryotes and eukaryotes. In particular, the DNA encoding F 1  or a portion thereof may be derived from bacteria and eukaryotic microorganisms such as yeast, other fungi and cell lines of higher organisms and be selected from the group consisting of the gene encoding the F 1  subunit beta or a portion thereof and various combinations of said gene or portion with the genes encoding the other F 1  subunits or portions thereof. The method can be used i.a. for optimizing the formation of biomass or a desired product by a cell by expressing different levels of uncoupled ATPase activity in the cell, incubating the cell on a suitable substrate, measuring the conversion rate of substrate into biomass or the desired product at each level of ATPase expression, and choosing a level of ATPase expression at which the conversion rate is optimized.

[0001] This invention relates to a method of improving the production of biomass or a desired product from a cell by inducing conversion of ATP to ADP without primary effects on other cellular metabolites or functions. The invention also relates to a method of optimizing the production of biomass or a desired product from a cell utilizing this first method. The desired product may for example be lactic acid produced by lactic acid bacteria and ethanol or carbondioxide produced by yeast.

BACKGROUND OF THE INVENTION

[0002] A wide range of microorganisms are used for the production of various organic compounds and heterologous proteins. One example hereof is the production of lactic acid and other organic compounds by the lactic acid group of bacteria, which results in the acidification and flavouring of dairy products, better known as cheese and yougurt production.

[0003] From the microorganism's point of view, the organic compounds which are excreted from the cells are often merely the by-product of a process that is vital to the cells: the production of various forms of free energy (ATP, NAD(P)H, membrane potential, etc.). Therefore, although many of the microorganisms which are being employed in these processes are reasonably well suited for the purpose, there is still a great potential for optimizing the productivity of these organisms when looking from the bioreactor point of vue. Likewise, the production of heterologous proteins by a microorganism is not what the organism was adapted for and also here there is a potential for optimization.

[0004] Often when microorganisms are engineered for the purpose of optimizing an industrial production process, the reactions leading to the desired product will affect the delicate balance of co-factors involved in the energy metabolism of the cell. For instance if the glycolytic reactions producing lactate from sugar were somehow to be enhanced (e.g. by overexpressing the glycolytic enzymes) this would automatically lead to the convertion of ADP to ATP. The ratio between the concentrations of ATP and ADP is usually quite high in the growing cell ([ATP]/[ADP]>10), and when the ratio [ATP]/[ADP] changes, the sum of [ATP] and [ADP] still remains virtually constant. Therefore, if in the example above, the enhanced production of ATP changes the [ATP]/[ADP] ratio from 10 to say 30, this will only marginally affect the concentration of ATP. The ADP concentration however will change by a factor of three. The cells will then hardly feel the surplus of ATP but the ADP pool in the cells may be depleted to such an extent that reactions in which ADP is a co-factor or allosteric regulator will be suppressed by the lack of ADP. The result may be that the total flux through the pathway (here through glycolysis) is only marginally increased. In the future, this situation is likely to occur more frequently, as the productivity of bioreactors are optimized by other means, and in these cases, it will be even more important (compared to the normal cell) to regenerate the ADP from ATP, in order to further increase the productivity.

[0005] Previously, attempts have been made to decrease the intracellular ATP concentration in yeast, employing sets of reactions which together form futile cycles, see EP patent No. 245 481. Often, the first reaction of a futile cycle is part of the regular metabolic network of the cell, for instance the phosphorylation of a glycolytic intermediate, coupled to the utilisation of ATP. The second reaction, which may also sometimes be part of the metabolic network, then de-phosphorylates the glycolytic intermediate without regenerating the ATP that was consumed in the first process, the overall effect being that a high energy phosphate bond is consumed. The limited success that this strategy has had so far, is probably due to the fact that it is impossible to obtain a significant futile flux without decreasing the concentration of the phosphorylated intermediate, thereby disturbing the cellular function and ultimately the growth. In addition, when the approach is to decrease the concentration of a glycolytic intermediate, this will effectively remove the substrate for the remaining part of the glycolysis, which will often result in a decreased flux through this pathway, rather than the desired increased flux.

[0006] Other strategies have been to use chemicals such as dinitrophosphate to stimulate the activity of the plasma membrane H⁺-ATPase by the addition of uncouplers of the membrane potential, or to genetically express the enzyme acid phosphatase in the cytoplasm, an enzyme that will remove phosphate groups from organic metabolites and proteins. However, both of these approaches suffer from the same inherent problem: they are unspecific and a range of cellular reactions/concentrations may be affected. For instance, the acid phosphatase will remove phosphate groups from essential metabolites and proteins, thus disturbing various metabolic fluxes and metabolic regulation. The uncoupling of the plasma membrane H⁺-ATPase will disturb the intracellular pH in addition to the gradient of numerous ions across the cytoplasmic membrane. Besides, the addition of chemicals such as dinitrophosphate is undesirable for most purposes.

SUMMARY OF THE INVENTION

[0007] The idea of the invention is to use a highly specific and clean way to increase the intracellular level of ADP, which does not suffer from the limitations described above: to express in a well-controlled manner an enzyme that has ATP-hydrolytic activity in the living cell without producing other products and without coupling this activity to energy conservation. Such an enzymatic activity is of course not likely to be found in a normal cell, because the cell would then loose some of its vital energy reservoir.

[0008] Accordingly the present invention provides a method of improving the production of biomass or a desired product from a cell, the method being characterized by expressing an uncoupled ATPase activity in said cell to induce conversion of ATP to ADP without primary effects on other cellular metabolites or functions, and incubating the cell with a suitable substrate to produce said biomass or product.

[0009] One of the normal enzymes that comes closest to the ideal ATP-hydrolyzing enzyme, is the membrane bound H⁺-ATPase. This huge enzyme complex consists of two parts, the membrane integral part (F₀) and the cytoplasmic part (F₁). Together the two parts couples the hydrolysis of ATP to ADP and inorganic phosphate (P_(i)), to translocation of protons accross the cytoplasmic membrane, or vice versa, using the proton gradient to drive ATP synthesis from ADP and P_(i).

[0010] The method of the invention is conveniently carried out by expressing in said cell the soluble part (F₁) of the membrane bound (F₀F₁ type) H⁺-ATPase or a portion of the F₁ exhibiting ATPase activity.

[0011] The membrane bound H⁺-ATPase complex is found in similar form in prokaryotic as well as eukaryotic organisms, and thus F₁ and portions thereof expressing ATPase activity can be expressed in both prokaryotic and eukaryotic cells.

[0012] The organism from which the F1 ATPase or portions thereof is derived, or in which the F1 ATPase or portions thereof is expressed, may be selected from prokaryotes and eukaryotes, in particular from bacteria and eukaryotic microorganisms such as yeasts, other fungi and cell lines of higher organisms, in particular bakers and brewers yeast.

[0013] A particularly interesting group of prokaryotes to which the method according to the invention can be implemented, i.a. in the dairy industry, are lactic acid bacteria of the genera Lactococcus, Streptococcus, Enterococcus, Lactobacillus and Leuconostoc, in particular strains of the species Lactococcus lactis and Streptococcus thermophilus. Other interesting prokaryotes are bacteria belonging to the genera Escherichia, Zymomonas, Bacillus and Pseudomonas, in particular the species Escherichia coli, Zymomonas mobilis, Bacillus subtilis and Pseudomonas putida.

[0014] In an expedient manner of carrying out the method according to the invention the cell is transformed or transfected with an expression vector including DNA encoding F₁ or a portion thereof exhibiting ATPase activity under the control of a promoter functioning in said cell, and said DNA is expressed in the cell. Said DNA encoding F₁ or a portion thereof may be derived from a prokaryotic or a eukaryotic organism, and it may be either homologous or heterologous to said cell.

[0015] The F₁ part of the bacterial H⁺-ATPase complex consists of several subunits that together are responsible for catalyzing ATP hydrolysis: the β-subunit is thought to carry the actual hydrolytic site for ATP hydrolysis, but in vitro ATPase activity requires that the β-subunit forms a complex together with the α- and γ-subunit (α₃γβ₃). The activity of this complex is modulated by the ε-subunit, so that the in vitro activity of the α₃γβ₃ε complex is five fold less than the α₃γβ₃ complex.

[0016] In a specific embodiment of the method according to the invention said DNA encoding F₁ or a catalytic active portion thereof, is derived from Escherichia coli, Streptococcus thermophilus or Lactococcus lactis and is selected from the group consisting of the gene encoding the F₁ subunit β or a catalytically active portion thereof and various combinations of said gene or portion with the genes encoding the F₁ subunits δ, α, γ and ε or catalytically active portions thereof.

[0017] In particular said DNA encoding F₁ or a portion thereof may be selected from the group consisting of the Escherichia coli, Streptococcus thermophilus and Lactococcus lactis genes atpHAGDC (coding for subunits δ, α, γ, β, ε), atpAGDC (coding for subunits α, γ, β, ε), atpAGD (coding for subunits α, γ, β), atpDC (coding for subunits β, ε) and atpD (coding for subunit β alone).

[0018] Particularly interesting eukaryotes are the yeasts Saccharomyces cerevisiae, Phaffia rhodozyma or Trichoderma reesei, and the DNA encoding F₁ or a portion thereof may be derived from such organisms and is selected from the group consisting of the gene encoding the F₁ subunit β or a portion thereof and various combinations of said gene or portion with the genes encoding the other F₁ subunits or portions thereof.

[0019] Vectors including DNA encoding the soluble part (F₁) of the membrane bound (F₀F₁ type) H⁺-ATPase or a portion of F₁ exhibiting ATPase activity, derived from the lactic acid bacteria Lactococcus lactis and Streptococcus thermophilus and from the yeasts Saccharomyces cerevisiae, Phaffia rhodozyma or Trichoderma reesei are also comprised by the invention as well as expression vectors including such DNA under the control of a promoter capable of directing the expression of said DNA in a prokaryotic or eukaryotic cell.

[0020] Specific vectors according to the invention are plasmids including DNA encoding the soluble part (F₁) of the membrane bound (F₀F₁ type) H⁺-ATPase or a portion of F₁ exhibiting ATPase activity, said DNA being derived from Lactococcus lactis subsp. cremoris (SEQ ID No. 1), Lactococcus lactis subsp. lactis (SEQ ID No. 6), Streptococcus thermophilus (SEQ ID No. 10), Phaffia rhodozyma (SEQ ID No. 14), and Trichoderma reesei (SEQ ID No. 16).

[0021] Further, the invention provides a method of optimizing the formation of biomass or a desired product by a cell, the method being characterized by expressing different levels of uncoupled ATPase activity in the cell, incubating the cell on a suitable substrate, measuring the conversion rate of substrate into biomass or the desired product at each level of ATPase expression, and choosing a level of ATPase expression at which the conversion rate is optimized.

[0022] Often, but not always, the optimization of a given product flux produced by a cell will entail the attainment of either maximum or minimum conversion rate of a substrate.

[0023] In an expedient manner of practicing this method of the invention a number of specimens of said cell are transformed or transfected with their respective expression vector each including DNA encoding a different portion of the cytoplasmic part (F₁) of the membrane bound (F₀F₁ type) H⁺-ATPase up to and including the entire F₁, each portion exhibiting ATPase activity, said DNA in each expression vector being under the control of a promoter functioning in said cell, incubating each cell specimen on a suitable substrate, measuring the conversion rate of substrate into biomass or the desired product in each specimen, and choosing a specimen yielding an optimal conversion rate. In a particular embodiment of this manner, which is especially suited for scientific studies, the promoter in each expression vector is an inducible promoter, and each cell specimen is grown at different concentrations of inducer in order to fine-tune the optimal conversion rate.

[0024] In a preferred manner of practicing the above method of optimizing the performance of a cell a number of specimens of said cell are transformed or transfected with their respective expression vector including DNA encoding a portion of the cytoplasmic part (F₁) of the membrane bound (F₀F₁ type) H⁺-ATPase up to and including the entire F₁, said portion exhibiting ATPase activity, said DNA in the respective expression vectors being under the control of each of a series of promoters covering a broad range of promoter activities and functioning in said cell, incubating each cell specimen on a suitable substrate, measuring the conversion rate of substrate into biomass or the desired product by each specimen, and choosing a specimen yielding an optimal conversion rate. In a more preferred embodiment of this manner, which is well suited to establish an optimal production strain, the respective expression vectors include DNA encoding different such portions of F₁ up to and including the entire F₁, each DNA in respective expression vectors being under the control of each of a series of promoters covering a broad range of promoter activities and functioning in said cell.

[0025] Also in this method of the invention the DNA encoding a portion of F₁ up to and including the entire F₁ may be derived from a prokaryotic or a eukaryotic organism, and it may be either homologous or heterologous to said organism. The specific DNAs mentioned above may also conveniently be employed in this method.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1. A linear representation of the plasmids constructed for modulating the cellular [ATP]/[ADP] ratio in E. coli (not drawn to scale).

[0027]FIG. 2. Effect of induction of F₁-ATPase activity on the growth of E. coli in batch culture. Cells were grown for more than 10 generations in minimal medium supplemented with glucose (0.4 g/l), ampicillin (0.1 g/l) and the indicated concentration of inducer, IPTG.

[0028]FIG. 3. Effect of ATPase expression on the intracellular concentration of ATP and ADP (concentration in arbitrary units), and on the ratio [ATP]/[ADP].

[0029]FIG. 4 Effect of increased ATPase expression on the glycolytic flux.

DETAILED DESCRIPTION OF THE INVENTION

[0030] Many biosynthetic reactions in the living cell (anabolism), require an input of free energy (ATP), which is generated through a series of degrading reactions (catabolism). In the aerobic cell, there are two routes for ATP synthesis: 1) substrate level phosphorylation, where an energy rich phosphoryl group is transferred directly from a high energy intermediate metabolite to ADP, and 2) oxidative phosphorylation, where the free energy is first transformed into redox free energy by oxidizing the energy source, then into a proton gradient by respiration and finally the proton gradient is used by the H⁺-ATPase to drive ATP synthesis from ADP and inorganic phosphate. In other cases, e.g. anaerobic growth, there is only the first route, substrate level phosphorylation, that can be used for ATP synthesis. An example hereof is the homolactic LAB, where lactose is converted through the glycolytic pathway to lactic acid, which is excreted from the cells and thereby lowers the pH of the growth medium (usually milk products). With respect to ATP generation, homolactic fermentation is a very inefficient process, and only four moles of ATP are produced from 1 mole of lactose through substrate level phosphorylation.

[0031] The anabolic (ATP consuming) and catabolic (ATP producing) fluxes are normally well balanced in the living cell, and therefore, in the wild-type cell under normal growth conditions, the catabolic fluxes will be proportional to the anabolic fluxes. If a reaction is introduced that for instance hydrolyzes ATP in the cell and thereby lowers the cellular energy state (i.e. the [ATP]/[ADP] ratio), then either catabolism should increase or anabolism (growth) should decrease in order to make the consumption rate equal the production rate again. Which of these two scenarios will take place depends on whether, initially, the growth rate of the cell is limited through anabolism or through catabolism, i.e. whether there is a surplus or a shortage of energy in the cell to begin with. If there is a shortage of energy, then the rate of the anabolic reactions is limited by catabolism and these reactions will be sensitive to changes in the cellular energy state. Introduction of an ATP-hydrolyzing reaction is then most likely to affect the growth rate of the cells. On the other hand, if there is a surplus of energy, then the growth rate will be limited mainly by the anabolic reactions; the rate of anabolism will be insensitive to a decrease in the energy state, but the catabolic rate may increase due to a decrease in product inhibition at lower [ATP]/[ADP] ratio.

[0032] In vitro, the F₁ part of the H⁺-ATPase complex has been shown to have ATPase actitity, see above. But so far nobody has managed to use the F₁ complex to stimulate the glycolytic flux, or even to show that the F₁ complex can hydrolyze ATP in intact cells. Indeed, when we first tried to overexpress the F1 complex, consisting of the genes for the subunits α, γ, β and ε, this had virtually no effect on the growth of E. coli, even when the genes were transcribed from the maximally induced tac promoter and on a very high copy number vector (derived from pUC18). One skilled in the art of gene expression in E. coli will appreciate that this combination is one of the most efficient expression systems that exists for this organism.

[0033] We then decided to try to express different combinations of subunits of the F1 complex, in order to see if other combinations of subunits would be more powerful. Plasmids were constructed containing various combinations of the genes encoding the F₁ part of the bacterial F₁F₀ -ATPase complex from E. coli. The genes were expressed, either from an inducible (lac-type) promoter at various concentrations of inducer or from a series of constitutive promoters of varying promoter activity. These plasmids should express various levels of ATPase activity when introduced into the bacterial cell. Depending on which F₁ genes are present on the plasmid and the strength of the promoter which is used to drive the expression, we observed various degrees of inhibition of the growth of the cells harbouring these plasmids. Surprisingly, the beta subunit alone and in combination with the epsilon subunit turned out to be far more active in vivo than the entire F1 complex.

[0034] The objective of this work was to affect the energy state of the cells, as reflected in the ratio [ATP]/[ADP]. We therefore measured the intracellular concentration of ATP and ADP in growing cells expressing various activities of F₁-ATPase. Indeed the ATP concentration decreased slightly with increasing ATPase activity and the ADP concentration increased, and therefore the [ATP]/[ADP] ratio decreased (the effect on the ATP concentration was less than the effect on the ADP concentration as expected, see above). We also calculated the glycolytic flux through the cells with various levels of ATPase activity. We found that the flux through the glycolytic pathway was first stimulated with increasing expression of ATPase activity, until a certain (optimal) ATPase activity which gave maximal glycolytic flux. Further increase of ATPase expression resulted in a lower glycolytic flux, due to a secondary effect of the ATPase activity on the growth of the cells. This emphasizes the need for optimization of gene expression rather than merely overexpressing the genes.

EXAMPLE 1 ATP Hydrolysis and Enhanced Glycolytic Flux in Escherichia coli, Using an Inducible Promoter

[0035] Restriction enzymes, T4 DNA polymerase, calf intestine phosphatase (CIP) were obtained from Pharmacia.

[0036] Procedures for DNA isolation, cutting with restriction enzymes, filling in sticky DNA ends with T4 DNA polymerase in the presence of dATP, dCTP, dGTP and dTTP, treatment with calf intestine phophatase to remove phosphate groups from 5′ DNA ends and ligation of DNA fragments are carried out by standard methods as described by Maniatis et al., 1982.

[0037] Extraction and Measurement of ATP and ADP

[0038] 0.9 ml of cell culture was mixed with 0.9 ml of (80° C.) phenol (equilibrated with 10 mM Tris, 1 mM EDTA pH=8) and immediately vortexed vigorously for 10 seconds. After 1 hour at room temperature the sample was vortexed again for 10 seconds and the two phases were separated by centrifugation at 14000 rpm for 15 minutes, and then residual phenol in the water phase was removed by extraction with 1 volume of chloroform. ATP and ADP concentrations were then measured, using, a luciferin-luciferase ATP monitoring kit (obtained from and used as recommended by LKB, except that 3 mM of phosphoenol-pyruvate was added). [ATP] was measured first. Subsequently the ADP in the same sample was converted to ATP by adding pyruvate kinase, and [ADP] was recorded as the concomitant increase in luminescence.

[0039] Construction of Plasmids Carrying Combinations of the E. coli atp Genes

[0040] The following combinations of E. coli genes coding for F₁ subunits were chosen for expressing ATPase activity in E. coli: 1. atpAGDC (subunits α, γ, β, ε), 2. atpAGD (subunits α, γ, β), 3. atpDC (subunits β, ε), and 4. atpD (subunit β alone).

[0041] Cloning of Fragments Carrying atp Genes onto pUC19

[0042] The plasmid pBJC917 (von Meyenburg, K., et al., 1984) which carries the entire atp operon was cut with

[0043] 1) the restriction enzyme DraIII, and a 5009 bp DNA fragment containing the atpAGDC genes was isolated;

[0044] 2) the restriction enzymes DraIII and Tth111I, and a 4106 bp DNA fragment containing the atpAGD genes was isolated;

[0045] 3) the restriction enzymes DraIII and SacII, and a 2364 bp DNA fragment containing the atpDC genes was isolated;

[0046] 4) the restriction enzymes AvaI and Tth111I, and a 1472 bp DNA fragment containing the atpD gene was isolated.

[0047] In all four cases the fragments were then treated with T4 DNA polymerase to create blunt ends, and subsequently the fragments were ligated into the cloning vector pUC19 (Yanisch-Perron et al.,1985) which had been cut with SmaI and treated with CIP.

[0048] The four ligation mixtures were transformed into the E. coli strain JM105 (Yanisch-Perron et al.,1985), and the transformation mixtures were plated on LB (Luria-Bertani broth; Maniatis et al., 1982) plates containing 100 μg/ml ampicillin and 75 μg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal). In this strain background (JM105), plasmids formed by religation of pUC19 will give blue colonies, whereas plasmids that carry foreign DNA fragments inserted into the SmaI site of pUC19, will give white colonies. A number of white colonies from the four transformations were therefore picked for further analysis: plasmid DNA was isolated and analysed by cutting with various restriction enzymes. Clones were identified from each of the four series which had the desired fragment inserted into the SmaI site of pUC19, and in the proper orientation. These four plasmids were named, respectively: pATP-AGDC, pATP-AGD, pATP-DC and pATP-D, with reference to the specific atp genes carried by the plasmid.

[0049] Cloning Combinations of the atp Genes Under the Control of an Inducible (tac) Promoter

[0050] In order to be able to control the expression of the ATPase activity, we selected the expression vector pTTQ18 (Starck, 1987). This vector is a derivative of PUC18 (Yanisch-Perron et al.,1985), which carries a tac promoter and the lactose repressor gene, lacI. Immediately downstream of the tac promoter is a multiple cloning site (MCS; the polylinker from pUC18) in which genes can be inserted to be expressed from the tac promoter. The tac promoter is of the lac-type, i.e. repressed by the lactose repressor and inducible with isopropyl-β-D-thiogalactoside (IPTG).

[0051] The four plasmids, pATP-AGDC, pATP-AGD, pATP-DC and pATP-D were cut with KpnI and XbaI, which gave the four DNA fragments, 5023, 4120, 2378 and 1486 respectively. After purification, the fragments were ligated into the cloning vector, pTTQ18, which had also been cut with KpnI and XbaI (see FIG. 1). The ligation mixtures were transformed into E. coli K-12 MC1000 (Casabadan and Cohen, 1980), and the transformation mixtures were plated on LB plates containing 100 μg/ml ampicillin. A number of colonies from the four transformations were therefore picked for further analysis: plasmid DNA was isolated and analysed by cutting with various restriction enzymes. Clones were identified from each of the four series which had the desired fragment inserted into the MCS of pTTQ18 in the proper orientation. These four plasmids were named, respectively: pTAC-AGDC, pTAC-AGD, pTAC-DC and pTAC-D, with reference to the specific atp genes carried by these plasmid and the tac promoter used for their expression. For the purpose of subsequent physiological studies, the plasmids were transformed into the E. coli K-12 strain LM3118, which is used routinely for physiological experiments in this laboratory. The corresponding names for the LM3118 strain carrying these four plasmids are PJ4332, PJ4333, PJ4335 and PJ4334, respectively.

[0052] Effect of Induction of ATPase Activity on the Growth of E. coli on Plates

[0053] The strains containing the four plasmids were streaked on LB plates containing ampicillin (100 μg/ml) and 1 mm of IPTG which should give maximum expression from the tac promoter. Table I shows how the four strains responded: the strain carrying plasmid pATP-AGDC, which contains the genes for the four subunits, α, γ, β and ε, was only very slightly affected in growth, even in the presence of 1 mM IPTG. The other three plasmids, pTAC-AGD, pTAC-DC and pTAC-D caused severe growth inhibition in the presence of 1 mM IPTG, where colonies were no longer visible. With intermediate concentrations of IPTG, 0.01 mM and 0.1 mM, the plasmids affected the growth of their host cells to different extents: pTAC-AGD was the most active, giving rise to a strong inhibition of growth already with 0.01 mM IPTG, a concentration which gave only a slight inhibition with the plasmid pTAC-DC and no inhibition of the strain with pTAC-D. With 0.1 mM IPTG, colonies were hardly visible for the strain that carried the pTAC-AGD, the plasmid pTAC-DC caused strong growth inhibition, whereas the effect of pTAC-D was significant but small. TABLE I 0.01 mM 0.1 mM 1 mM Strain Plasmid − IPTG IPTG IPTG IPTG PJ4332 pTAC-AGDC ++++ ++++ ++++ +++ PJ4333 pTAC-AGD ++++ ++ + − PJ4335 pTAC-DC ++++ +++ + − PJ4334 pTAC-D ++++ ++++ ++ −

[0054] The effect of ATPase expression from the four plasmids above was also studied in the E. coli mutant LM3115, in which the entire atp operon on the chromosome is deleted, but which grows with almost wild-type growth rate on LB medium. With this strain transformed with the four plasmids we observed a similar pattern of growth inhibition on LB plates as a function of IPTG concentration. This shows that the effect of ATPase expression was independent of the presence of the normal atp operon.

[0055] Effect of Induction of ATPase Activity on the Growth of E. coli in Liquid Cultures

[0056] The effect of induction of ATPase was also studied with cells grown in liquid cultures. For this purpose we chose the strain PJ4333, carrying the plasmid pTAC-AGD, because this plasmid appears to be the most active with respect to the inhibitory effect on the of growth of E. coli. FIG. 2 shows the growth of PJ4333 in minimal medium supplemented with a limiting concentration of glucose (0.4 g/l) and ampicillin (0.1 g/l), without IPTG and in the presence of increasing concentrations of IPTG. We observed that the growth rate of the strain was practically constant (within some 10%) with increasing amounts of IPTG up to about 30 μM. At higher than 40 μM IPTG, the growth of the cells were slightly inhibited, in accordance with the experiments on plates, see above.

[0057] However, what was affected was the final density of cells that one obtains from the limited amount of glucose that was included in each culture: The more ATPase that is expressed in the cells, the lower the yield of cell mass. Apparently, the cells become less economic with respect to converting the glucose into biomass, or in other words they consume more glucose per cell synthesized. If this is due to the expression of ATPase activity, then we would expect to see an effect hereof on the energy state of the cells. We therefore measured the concentrations of ATP and ADP in the cells growing with different expression levels of ATPase activity.

[0058] Indeed, the intracellular ATP concentration decreased gradually and the ADP concentration increased, with increased expression of ATPase; therefore the [ATP]/[ADP] ratio decreased with increased expression of ATPase, which imply that the increased glucose consumption is the result of increased ATP convertion to ADP, see FIG. 3. The actual flux of glucose through the cells (J_(gluc), mmol glucose/g cell dry weight/hour) is also interesting, because this value tells us whether the performance of the cell increased as the ATPase activity increased. J_(gluc) can be calculated from the yield, Y (g cell dry weight/mol glucose) and the specific growth rate of the culture, μ (1/hours):

J _(gluc) =μ/Y

[0059]FIG. 4 shows how the flux of glucose changed as the activity of ATPase increased: the glycolytic flux increased gradually as the ATPase expression increased, until a maximum was reached (at 30 μM IPTG). Further increase of ATPase expression had a slightly negative effect on the glucose flux. This was probably because the energy state of the cells became so low that this had a negative effect on some anabolic reactions, since the growth rate was lower for the culture that was grown in the presence of 40 μM IPTG.

[0060] The expression of subunits of the F₁ part of the bacterial H⁺-ATPase lowers the energy state of the bacterial cell. This is due to hydrolysis of ATP into ADP and P_(i). The expression of ATPase activity does not affect the growth rate of E. coli much at low levels of expression, but the efficiency by which the substrate is converted into biomass was strongly reduced. Under the set of conditions used here, the expression of ATPase activity has a stimulatory effect on the rate by which the cells consumes the exogenous glucose.

EXAMPLE 2 Expression of F₁-ATPase Activity from Constitutive Promoters in E. coli

[0061] In example 1 we used a lac-type promoter system to modulate the expression of the F₁ ATPase subunits in E. coli. However, for the optimization of gene expression for instance in industrial bioreactors or for the use in fermented food products, the use of lac type promoters is not always feasible. In this example we illustate the optimization of F₁-ATPase expression in E. coli, using a series of constitutive promoters of different strength, to control the expression of the atpAGD genes which here originates from E. coli. The constitutive promoters (CP promoters) were selected from a library of artificial promoters which had previously been cloned onto a shuttle vector for E. coli and L. lactis, pAK80 (Israelsen et al., 1995) as described in our co-pending PCT patent application PCT/DK97/00342. The selected plasmid derivatives of pAK80 were pCP34, pCP41 and CP44 (CPX cloning vectors). The atpAGD fragment from pTAC-AGD (from example 1) was first subcloned in a polylinker in order to have the atpAGD fragment flanked by two BamHI sites. Subsequently, this BamHI fragment was cloned into the unique BamHI site downstream of the CP promoters on the plasmids pCP34, pCP41 and CP44, resulting in the plasmids, pCP34::atpAGD, pCP34::2atpAGD, pCP41::atpAGD and CP44::atpAGD, where pCP34::2atpAGD contains two atpAGD fragments in tandem.

[0062] Subsequently, the strains were characterized with respect to growth rate, growth yield and glycolytic flux in glucose minimal medium supplemented with 200 μg/ml erythromycin, essentially as described in example 1, see table 2.

[0063] The expression of the F₁-ATPase subunits had a slightly negative effect on the growth rate as the expresion level increased. The effect on growth yield was much stronger and at the highest expression level the growth yield had dropped to 40% of the initial value. The glycolytic flux was stimulated 70% at the highest expression level of ATPase, and at this expression level the growth rate was lowered by 30%. TABLE 2 Effect of expression of uncoupled F₁-ATPase activity (E. coli α, γ, β subunits) in E. coli Glucose Biomass flux Bio- yield Growth mmol mass Growth Glu- gdw/mmol rate, μ glucose/ yield rate cose Plasmid glucose h−1 h/gdw % % flux pCP41 0.067 0.47 6.9 100 100 100 pCP41::atpAGD 0.047 0.42 9.1 69 90 131 pCP34 0.063 0.41 6.6 100 100 100 pCP34::atpAGD 0.034 0.34 9.9 54 81 149 pCP44 0.067 0.44 6.5 100 100 100 pCP44::atpAGD 0.027 0.30 11.2 40 69 172

EXAMPLE 3 Expression of E. coli F₁-ATPase Activity from Constitutive Promoters in L. lactis

[0064] The plasmids from example 2 which express the E. coli F₁-ATPase subunits to various extent are also capable of replicating in L. lactis, and could therefore be used to test whether the E. coli F₁-ATPase subunits can be used to hydrolyse ATP in L. lactis.

[0065] The plasmids pCP34::atpAGD, pCP34::2atpAGD and pCP41::atpAGD, were transformed into the L. lactis subspecies cremoris strain, MG1363, which is used routinely for physiological experiments in this laboratory. In addition we transformed the respective vectors, pCP34 and pCP41 in order to have proper control strains. Subsequently, the resulting transformants were characterized with respect to growth rate, growth yield and glycolytic flux, in comparison to the respective vectors, pCP34 arid pCP41, by growing the various cultures in defined medium (SA medium) supplemented with a limiting concentration of glucose (0.1%), see table 3. TABLE 3 Expression of E. coli F₁-ATPase in L. lactis Glucose Biomass flux Bio- Glu- yield Growth mmol/ mass Growth cose gdw/mmol rate, μ glucose/ yield rate flux Plasmid glucose h−1 h/gdw % % % pCP34 0.073 0.664 9.161 100 100 100 pCP34::atpAGD 0.071 0.653 9.230 97.5 98.3 100.8 pCP34::2atpAGD 0.069 0.655 9.560 94.6 98.7 104.4 pCP41 0.072 0.645 8.925 100 100 100 pCP41::atpAGD 0.070 0.590 8.461 96.5 91.5 94.8

[0066] The results show that the plasmids pCP34::atpAGD and pCP34::2atpAGD did affect the growth yield and the glycolytic flux to some extent, but the plasmids were far less efficient in L. lactis, compared to E. coli. This was probably a consequence of a lower expression of the E. coli ATPase subunits, or some of these, in L. lactis, due to a lower copy number of the pAK80 vector in L. lactis (5-10), and due to differences in the translational effciency of the three individual atp genes which originates from E. coli. The plasmid pCP41::atpAGD also resulted in a lower growth yield, indicating that also in this case uncoupled ATP hydrolysis was taking place. However, the pCP41::atpAGD plasmid had a relatively strong inhibitory effect on the growth rate and therefore the glycolytic flux was not increased by this plasmid. It is possible that the heterologous expression of E. coli ATPase subunits resulted in growth inhibition due to effects other than ATP hydrolysis, e.g. by interfering with the function of the L. lactis F₁F₀ H⁺-ATPase complex.

EXAMPLE 4 Expression of L. lactis F₁-ATPase Subunits β and ε, in L. lactis

[0067] In the example above we showed that the expression of F₁-ATPase subunits from E. coli in L. lactis, resulted in only a small stimulation of the glycolytic flux. It is possible that the heterologous expression of E. coli ATPase subunits resulted in growth inhibition due to effects other than ATP hydrolysis, e.g. by interfering with the function of the L. lactis F₁F₀ H⁺-ATPase complex. In the present example we have expressed the L. lactis F₁-ATPase subunits, β and ε, in L. lactis, as this appeared to be an effective combination of subunits when expressed in E. coli, see example 1. The atpDC_(Llc) genes from L. lactis subspecies cremoris (SEQ ID No. 1) was cloned on a 2.5 kb HindIII fragment into the HindIII restriction site on the standard cloning vector, pBluescript, into E. coli K-12, strain BOE270. Subsequently, the atpDC_(Llc) genes were cut out on a 2.5 kb BamHI-SalI fragment and cloned into 5 expression vectors, pCP32, pCP34, pCP37, pCP41 and pCP44 which had been digested with BamHI and SalI, resulting in the plasmids pCP32::atpDC_(Llc), pCP34::atPDC_(Llc), pCP37::atpDC_(Llc), pCP41::atpDC_(Llc) and pCP44::atpDC_(Llc), respectively, where the lacLM genes downstream of the CP promoters, have been replaced with the atpDC_(Llc) genes. These plasmids should express the L. lactis F₁-ATPase subunits, β and ε, to different extent. The plasmids were then transformed into MG1363 with selection for the erythromycin resistance carried by these vectors. Experiments were then performed to test whether the constructs resulted in convertion of ATP into ADP in L. lactis. The strains carrying the different constructs was then grown in GM17 medium supplemented with 5 μg/ml erythromycin. The plasmids did not have a strong effect on the growth rate of the cultures, which remained close to the growth rate of the respective vector control plasmids. The yield of biomass, however, decreases for all the cultures by up to 17%, which shows that the cultures did indeed express uncoupled ATPase activity, see table 4. TABLE 4 Effect of expression of L. lactis β and ε subunits on acid production by L. lactis, at 30° C. and with initial pH 6.7. Acid formation, Biomass* relative to biomass* Plasmid OD₄₅₀ Final pH* % of vector pCP34 5.08 4.27 100 pCP34::atpDC11c 4.72 4.31 98.0 pCP41 4.66 4.34 100 pCP41::atpDC11c 5.21 4.24 113.5 pCP37 4.89 4.28 100 pCP37::atpDC11c 4.63 4.24 116.1 pCP32 4.86 4.34 100 pCP32::atpDC11c 3.95 4.36 116.9

[0068] The GM17 growth medium used in these experiments contains a surplus of glucose (1%), and growth only stops when the pH of the growth medium becomes lower than approximately pH 4.3. To some extent, this mimics the situation that the lactic acid bacteria experience during cheese and yougurt production. In this medium, the growth yield, in terms of the final cell mass of the cultures, reflects the acid production by these cultures.

[0069] In these cultures, the expression of F₁-ATPase subunits will increase three fold at approximately OD600 equal to 1.5. This is a consequence of the three fold amplification of the plasmid copy number that has been shown to take place at this point of the growth curve. In reality, the effect of expressing the F₁-ATPase subunits may therefore be larger.

[0070] To test this hypothesis, we grew some of the strains which expressed the L. lactis F₁-ATPase subunits β and ε in batch cultures of GM17 medium which had been adjusted to pH 5.9, see Table 5. In addition, the temperature of the growth medium may also affect the plasmid copy number and thus the expression of the F₁-ATPase subunits. The experiments were therefore performed at 37° C. TABLE 5 Effect of expression of L. lactis β and ε subunits on acid production by L. lactis, at 37° C. and with initial pH 5.9. Acid formation, Biomass* relative to biomass* Plasmid OD₄₅₀ Ffinal pH* % of vector pCP34 1.24 4.95 100 pCP34::atpDC_(11c) 1.06 4.87 141.4 pCP37 1.00 4.96 100 pCP37::atpDC_(11c) 0.58 4.92 188.4

[0071] Clearly, the effect of the F₁-ATPase activity was much stronger under these growth conditions: the amount of acid produced was almost doubled for the strain carrying the plasmid pCP37::atpDC_(llc).

EXAMPLE 5 Expression of the F₁-ATPase Subunits, α, γ, and β, from L. lactis Subspecies cremoris in L. lactis Subspecies cremoris

[0072] In example 4, only the L. lactis F₁-ATPase β and ε subunits were expressed in L. lactis. However, from the experiments with E. coli (example 1), we know that the simultaneous expression of subunits α, γ, and β, is a more powerful combination, which could also be the case for L. lactis. In order to obtain the same strong stimulation of the glycolytic flux and acid production in L. lactis, a set of vectors similar to the vectors described in example 4 was constructed, in which the atpAGD_(Llc) genes derived from L. lactis, encoding the subunits α, γ, and β (SEQ ID No. 1) was expressed from CP promoters with different activities. The atpAGD_(Llc) genes from L. lactis was cloned on a 2.5 kb BamHI-SalI fragment into the 5 vectors, pCP32, pCP34, pCP37, pCP41 and pCP44, resulting in the plasmids, pCP32::atpAGD_(Llc), pCP34::atpAGD_(Llc), pCP37::atpAGD_(Llc), pCP41::atpAGD_(Llc), pCP44::atpAGD_(Llc), respectively, where the lacLM genes downstream of the CP promoters, has been replaced with the atpAGD_(Llc) genes. These plasmids will express the L. lactis F₁-ATPase subunits α, γ, and β, to different extent. The plasmids were transformed into MG1363 with selection for the Erythromycin resistance carried by these vectors. Experiments were then performed to show that the constructs were effective in ATP hydrolysis in L. lactis and to what extent the glycolytic flux was enhanced, by growing the five different constructs in GM17 medium supplemented with erythromycin, and measuring the growth rate, ATP and ADP concentrations, the yield of biomass and the rate of acid production.

EXAMPLE 6 Expression of F₁-ATPase Subunits from L. lactis Subsp lactis, in L. lactis Subspecies lactis

[0073] In the examples 3-5 above, we used the strain L. lactis subsp. cremoris, MG1363. This strain is plasmid-free and is used routinely in our laboratory as a simple model organism for our physiological studies. But strains belonging to the subspecies lactis are also important in cheese production. We therefore cloned and sequenced the atpAGD_(Lll) genes from L. lactis subsp. lactis, (SEQ ID No. 6). Subsequently, a 4.2 kb fragment habouring the atpAGD_(Lll) genes was cloned into 5 vectors, pCP32, pCP34, pCP37, pCP41 and pCP44, resulting in the plasmids, pCP32::atpAGD_(Lll), pCP34::atpAGD_(Lll), pCP37::atpAGD_(Lll), pCP41::atpAGD_(Lll), pCP44::atpAGD_(Lll), respectively. These plasmids were then transformed into L. lactis subsp. lactis as described in example 3. The resulting strains with different expression levels of the F₁-ATPase subunits α, γ and β were then used to characterize the effect hereof on the growth yield, growth rate, glycolytic flux, and the cellular energy state of L. lactis subsp. lactis, as described in the examples 1-5.

EXAMPLE 7 Expression of F1-ATPase Subunits from S. thermophilus, ST3, in S. thermophilus, ST3

[0074] In the examples 3-6 above, we used strains of the genus Lactococcus. These strains are important in cheese production. As starter cultures for yougurt production, the dairy industry often uses strains of S. thermophilus. We therefore cloned and sequenced the atpAGD_(St) genes from S. thermophilus, strain ST3 (SEQ ID No. 10). Subsequently, a 4.2 kb fragment habouring the atpAGD_(St) genes was cloned into the 5 vectors, pCP32, pCP34, pCP37, pCP41 and pCP44, resulting in the plasmids, pCP32::atpAGD_(St), pCP34::atpAGD_(St), pCP37::atpAGD_(St), pCP41::atpAGD_(St), pCP44::atpAGD_(St), respectively. These plasmids were then transformed into S. thermophilus strain ST3. The resulting strains have different expression levels of the F₁-ATPase subunits α, γ, and β, and were then used to characterize the effect hereof on the growth yield, growth rate, glycolytic flux, and the cellular energy state of S. thermophilus, as described in the previous examples.

EXAMPLE 8 Expression of a Truncated F₁-ATPase β Subunit from Phaffia rhodozyma in Saccharomyces Cerevisiae

[0075] In this example we show that uncoupled F₁-ATPase expression can also be used to hydrolyze ATP in yeast cells of Saccharomyces cerevisiae.

[0076] A cDNA gene library was prepared from total RNA, isolated from Phaffia rhodozyma, by cloning the cDNA fragments into the expression vector, pYES2.0. One of the resulting plasmids, pATPbeta, gave rise to an ade⁺ phenotype in the Saccharomyces cerevisiae strain, W301, which carries a mutation in the ADE2 gene. Sequencing of the clone revealed a 0.9 kb insert, which encoded a protein of 254 amino acids (SEQ ID No. 14). The encoded protein had a very high homology to the C-terminal part of F₁-ATPase β subunits from other organisms, prokaryotic as well as eukaryotic, including the β subunit from S. cerevisiae (86% identity).

[0077] The ADE2 mutation results in starvation for an intermediate further down in the purine metabolism, AICAR (which under normal conditions is produced by ADE3, two steps further down in this pathway). AICAR is essential for de novo biosynthesis of AMP and GMP, and ADE2 mutants therefore need an alternative purine source in the growth medium. However, there is an alternative route for synthesis of AICAR which involves some of the genes involved in histidine biosynthesis. These genes are normally repressed under the conditions used for the complementation, but when the HIS3 gene is introduced on a plasmid, this complements the ADE2 mutation because the cells start to produce AICAR. Since AICAR is a precursor for ATP, it is likely that a lack of ATP (or increased levels of ADP and AMP) provide a signal to derepress the HIS3 gene and generate AICAR (which will subsequently end up as ATP). Indeed, cross-pathway regulation between purine and histidine biosynthesis has been found in yeast and involves the transcription factors BAS1 and BAS2. A reasonable explanation for the ade⁺ phenotype conferred by the plasmid, is therefore that the plasmid gives rise to ATP hydrolysis in the cytoplasm, thereby effecting the concentrations of adenine nucleotides in the cytoplasm.

[0078] Importantly, this truncated β subunit from Phaffia rhodozyma that was encoded on pATPbeta, included the region of the β subunit which is thought to encode the catalytic site for ATP hydrolysis. The truncation of the N-terminal part of the β subunit probably means that the protein will no longer be exported into the mitochondrion, but should stay within the yeast cytoplasm.

[0079] The truncated β subunit pATPbeta is expressed from a gal promoter, i.e. it can be induced with galactose. If the truncated β subunit encoded by the clone is active in ATP hydrolysis it should result in a decrease in the growth yield, and at sufficiently high expression level, we should also observe inhibition of growth. The strain which expressed the truncated β subunit and a control strain (which contained a plasmid pHIS3 containing a HIS3 gene from Phaffia rhodozyma), were streaked on plates containing galactose as the energy source, whicn will give maximal expression of the truncated β subunit. Indeed, the growth of the strain which expressed the truncated β subunit was strongly inhibited by the presence of galactose, whereas the control strain grew normally. As a control, the growth of the two strains were also compared on a plate containing glucose as the energy source, conditions under which the expression of the β subunit should be repressed, and indeed we observed little difference in growth of the two strains on these plates, see table 6.

[0080] Subsequently, for the purpose of the physiological investigations, the two strains were converted into Rho⁻ strains (petit mutants, defective in oxidative phosphorylation) by standard treatment with ethidium bromide. The induction with galactose caused even stronger inhibition of growth in the Rho⁻ background, which further indicates that the cause of the growth inhibition is uncoupled ATP hydrolysis in the cytoplasm. TABLE 6 Effect of expression of a truncated F₁-ATPase β subunit from Phaffia rhodozyma in S. cerevisiae on SC plates Strain/plasmid SC-ura + glucose SC-ura + galactose Rho⁺/pATPbeta +++++ + Rho⁺/pHIS3 ++++ +++ Rho⁻/pATPbeta +++++ − Rho⁻/pHIS3 ++++ +++

[0081] Growth experiments were performed to measure the resulting changes in the ATP/ADP ratio and the degree of stimulation of the glycolytic flux and ethanol formation, essentially as described in the examples above, and to show that the truncated β subunit from Phaffia rhodozyma is active with respect to converting ATP into ADP in the yeast cell.

EXAMPLE 9 Expression of F₁-ATPase β Subunit from Trichoderma reesei in Saccharomyces cerevisiae

[0082] In this example we show that the expression of the F₁-ATPase β subunit from the filamentous fungus, Trichoderma reesei can be used to improve the product formation of Saccharomyces cerevisiae.

[0083] The gene encoding the F₁-ATPase β subunit homologue from Trichoderma reesei was isolated from a cDNA library, inserted into a multicopy expression vector, pAJ401. DNA sequencing (SEQ ID 16) revealed that the cloned gene had very high homology to the β subunits from Neurospora crassa (91% identity), Kluyveromyces lactis (68%) and Saccharomyces cerevisiae (68%). Importantly, the first 43 amino acids in this β subunit, which encodes the signal for exporting the protein into the mitochondria, was homologous to the N-terminal part of the β subunit from Neurospora crassa (58% identity), but not to that of Saccharomyces cerevisiae. It is therefore likely that the β subunit from Trichoderma reesei will stay within the cytoplasm when expressed in Saccharomyces cerevisiae. This is important for the many cases where the fermentation is carried out anaerobically, because in these cases it is probably most efficient if the ATP hydrolysis takes place in the cytoplasm. Alternatively, in those cases where the β subunit is transported into the mitochondrion, it may be useful to genetically modify the β subunit so that is stays within the cytoplasm.

[0084] The gene encoding the F₁-ATPase β subunit homologue from Trichoderma reesei was expressed in S. cerevisiae strain VW1b (MAT alpha, leu2-3/112, ura3-52, trp1-289, his3D1, MAL2-8c, SUC2). To test whether the presence of the T. reesei β subunit resulted in ATP hydrolysis in the cytoplasm of the Saccharomyces cerevisiae host cells, we measured the intracellular concentrations of ATP, ADP and AMP, under various growth conditions in cultures of two strains expressing the β subunit (pATPβ34 and pATPβ44) and a strain carrying the vector plasmid, pFL60, see table 7. TABLE 7 Effect of expression of T. reesei β subunit on ATP, ADP and AMP concentrations* in S. cerevisiae ATP ADP AMP Strain μmol/gdw μmol/gdw μmol/gdw ATP/ADP ratio Aerobic/exp. phase pATPβ34 19.3 5.58 3.31 3.5 pATPβ44 13.9 5.15 3.25 2.7 pVECTOR 16.6 5.47 3.43 3.0 Aerobic/stat. phase pATPβ34 9.30 4.03 2.89 2.3 pATPβ44 8.99 3.90 2.42 2.3 pVECTOR 19.5 4.62 2.87 4.2 anaerobic/stat. phase pATPβ34 4.39 11.6 6.72 0.4 pATPβ44 3.14 10.5 6.65 0.3 pVECTOR 8.84 10.2 6.37 0.9

[0085] The β subunit did not appear to have a significant effect on the concentrations of ATP, ADP and AMP in cells growing on glucose in the exponential growth phase. The reason is probably that the ATP concentration that the homeostatic control of ATP synthesis can here keep up with the extra drain on ATP conferred by the β subunit F1-ATPase activity. Indeed, the growth rate of these cultures was unaffected by the presence of the F₁-ATPase activity, see table 7. But in the stationary cultures the concentration of ATP decreased significantly in the cultures expressing the β subunit, compared to the control. The effect was strongest in the anaerobically grown cultures where the ATP was lowered by a factor of 2-3. In these cultures, ATP must be generated through oxidative phosphorylation, (which is not even an option for the anaerobic cultures), and any effect of uncoupled ATP hydrolysis should therefore indeed be stronger in these cells.

[0086] Shake Flask Cultivations of Cultures Expressing the F₁-ATPase β Subunit Homologue in Saccharomyces cerevisiae.

[0087] Shake flask cultivations were performed under microaero-bic/anaerobic conditions with volume ratio 1/1.25 and no agitation; with 400 ml growth media in 500 ml Erlenmeyers on magnetic stirring. The growth media contained 5 g/l of glucose and amino acids and bases according to synthetic complete medium (SC−ura+0.5%G). OD₆₀₀ was monitored during the cultivation (OD600=1.0 is equal to 0.3 g/l dry weight). Ethanol and glucose were measured with HPLC (Waters, Sugar-Pak or IC-Pak columns). Production of ethanol (grams of ethanol per grams of cell dry weight) is shown in Table 8. TABLE 8 Effect of expression of T. reesei β subunit, on fluxes of ethanol and glucose in S. cerevisiae J_(gluc) J_(etoh) μ J_(gluc) J_(etoh) relative relative Strain h⁻¹ g/h/gdw g/h/gdw to control to control pATPβ34 0.40 2.811 1.190 107.7 105.6 pATPβ44 0.40 2.750 1.187 105.3 105.3 pVECTOR 0.39 2.611 1.127 100 100 control

[0088] These data show that the presence of the T. reesei F₁-ATPase β subunit resulted in an increased flux of glucose, as well as ethanol, in the Saccharomyces cerevisiae host cells.

REFERENCES

[0089] Casabadan, M. J., and Cohen, S. N. (1980). J. Mol. Biol., 138, 179-207.

[0090] Israelsen, H. (1995). Cloning and partial characterization of regulated promoters from Lactococcus lactis Tn917-lacZ integrants with the new promoter probe vector, pAK80. Appl. Environ. Microbiol., 61, 2540-2547.

[0091] Maniatis, T., Fritsch, E. F., and Sambrook, J., (1982). Molecular cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

[0092] Miller, J. H., (1972). Experiments in molecular genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

[0093] Starck, M. J. R. (1987). Multicopy expression vectors carrying the lac repressor gene for regulated high-level expression of genes in Escherichia coli. Gene 51, 255-267.

[0094] von Meyenburg, K., Joergensen, B. B., and van Deurs, B. (1984). Physiological and morphological effects of over-production of membrane-bound ATP synthase in Escherichia coli K-12. EMBO J. 3, 1791-1797.

[0095] Yanisch-Perron, C., Vieira, J., and Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103-109.

1 17 4815 base pairs nucleic acid double linear DNA (genomic) NO NO Lactococcus lactis subsp. cremoris MG1363 CDS 26..550 /codon_start= 26 /product= “ATPase subunit” /gene= “atpH” /standard_name= “delta subunit of the F1 portion of the F0F1 ATPase” /label= delta-subunit CDS 742..2241 /codon_start= 742 /product= “ATPase subunit” /gene= “atpA” /standard_name= “alpha subunit of the F1 portion of the F0F1 ATPase” /label= alpha-subunit CDS 2260..3126 /codon_start= 2260 /product= “ATPase subunit” /gene= “atpG” /standard_name= “gamma subunit of the F1 portion of the F0F1 ATPase” /label= gamma-subunit CDS 3301..4707 /codon_start= 3301 /product= “ATPase subunit” /gene= “atpD” /standard_name= “beta subunit of the F1 portion of the F0F1 ATPase” /label= beta-subunit 1 TATCTCGCTA AGTTAGGAGA ATAAG ATG ACA AAA GTA AAT TCA CAA AAA TAC 52 Met Thr Lys Val Asn Ser Gln Lys Tyr 1 5 AGT AAA GCT TTA CTT GAG GTC GCC CGA GAA AAA GGA CAA CTT GAA GCA 100 Ser Lys Ala Leu Leu Glu Val Ala Arg Glu Lys Gly Gln Leu Glu Ala 10 15 20 25 ATT CTT ACT GAA GTT AGC GAA ATG ATT CAG CTT TTC AAA GAA AAT AAC 148 Ile Leu Thr Glu Val Ser Glu Met Ile Gln Leu Phe Lys Glu Asn Asn 30 35 40 TTA GGT GCT TTT TTA GCA AAT GAA GTT TAT TCA TTC TCT GCT AAA TCT 196 Leu Gly Ala Phe Leu Ala Asn Glu Val Tyr Ser Phe Ser Ala Lys Ser 45 50 55 GAA TTG ATT GAT ACT TTG CTT CAA ACT TCA TCA GAA GTG ATG TCA AAT 244 Glu Leu Ile Asp Thr Leu Leu Gln Thr Ser Ser Glu Val Met Ser Asn 60 65 70 TTC CTG AAT ACT ATT CGT TCT AAT GGA CGT CTA GCT GAC CTC GGA GAA 292 Phe Leu Asn Thr Ile Arg Ser Asn Gly Arg Leu Ala Asp Leu Gly Glu 75 80 85 ATA CTT GAA GAA ACT AAA AAT GCA GCA GAT GAC ATG TTC AAA ATT GCT 340 Ile Leu Glu Glu Thr Lys Asn Ala Ala Asp Asp Met Phe Lys Ile Ala 90 95 100 105 GAC GTT GAA GTT GTT TCA AGT ATT GCA TTG TCA GAA GCT CAA ATT GAA 388 Asp Val Glu Val Val Ser Ser Ile Ala Leu Ser Glu Ala Gln Ile Glu 110 115 120 AAA TTT AAA GCA ATG GCT AAA TCA AAA TTT GAT TTA AAC GAA GTA ACA 436 Lys Phe Lys Ala Met Ala Lys Ser Lys Phe Asp Leu Asn Glu Val Thr 125 130 135 GTA ATT AAT ACA GTC AAT GAA AAA ATT CTC GGA GGA TTC ATT GTG AAC 484 Val Ile Asn Thr Val Asn Glu Lys Ile Leu Gly Gly Phe Ile Val Asn 140 145 150 TCT CGT GGA AAA ATT ATT GAC GCC TCA TTA AAA ACA CAA TTG GCT AAA 532 Ser Arg Gly Lys Ile Ile Asp Ala Ser Leu Lys Thr Gln Leu Ala Lys 155 160 165 ATC GCC GCT GAA ATC CTC TAATCAGGAT AGAAAAATTT TCTTCCTTTG 580 Ile Ala Ala Glu Ile Leu 170 175 TTAAAAACTT AGTGGAGAAT TTTTCAAACT CAAACTGTTA AACTTTTGAA AACATGCAAA 640 GGTAATTTTA AAACTTGCTT ATTCATGCTC AAAAAGTATA ACTGCAGTTT AAAGCTAAAT 700 AGCCTTGAAC TAGTAAAAAA TTTCTAGAAG GGAGCATATT T TTG GCA ATT AAA 753 Leu Ala Ile Lys 1 GCT AAT GAA ATC AGC TCA CTG ATT AAA AAA CAA ATT GAA AAT TTC ACA 801 Ala Asn Glu Ile Ser Ser Leu Ile Lys Lys Gln Ile Glu Asn Phe Thr 5 10 15 20 CCA GAT TTT GAA GTT GCT GAA ACT GGT GTC GTT ACC TAT GTT GGT GAT 849 Pro Asp Phe Glu Val Ala Glu Thr Gly Val Val Thr Tyr Val Gly Asp 25 30 35 GGT ATC GCG CGT GCC TAT GGC CTT GAA AAT GCG ATG AGC GGT GAG CTT 897 Gly Ile Ala Arg Ala Tyr Gly Leu Glu Asn Ala Met Ser Gly Glu Leu 40 45 50 GTT GAG TTT TCA AAT GGT ATA CTT GGT ATG GCG CAA AAC TTG GAT GCT 945 Val Glu Phe Ser Asn Gly Ile Leu Gly Met Ala Gln Asn Leu Asp Ala 55 60 65 ACA GAC GTT GGT ATT ATC GTA CTT GGT GAT TTC CTC TCA ATT CGT GAA 993 Thr Asp Val Gly Ile Ile Val Leu Gly Asp Phe Leu Ser Ile Arg Glu 70 75 80 GGT GAC ACT GTT AAA CGT ACA GGT AAA ATC ATG GAA ATC CAA GTT GGT 1041 Gly Asp Thr Val Lys Arg Thr Gly Lys Ile Met Glu Ile Gln Val Gly 85 90 95 100 GAA GAA CTC ATC GGA CGT GTT GTA AAC CCA CTT GGA CAA CCC GTT GAT 1089 Glu Glu Leu Ile Gly Arg Val Val Asn Pro Leu Gly Gln Pro Val Asp 105 110 115 GGA CTT GGA GAA CTT AAT ACA GGT AAA ACT CGT CCA GTT GAA GCA AAA 1137 Gly Leu Gly Glu Leu Asn Thr Gly Lys Thr Arg Pro Val Glu Ala Lys 120 125 130 GCT CCT GGT GTT ATG CAA CGT AAA TCA GTC TCT GAG CCA TTA CAA ACT 1185 Ala Pro Gly Val Met Gln Arg Lys Ser Val Ser Glu Pro Leu Gln Thr 135 140 145 GGT CTT AAA GCG ATT GAT GCC CTC GTT CCA ATT GGA CGT GGA CAA CGT 1233 Gly Leu Lys Ala Ile Asp Ala Leu Val Pro Ile Gly Arg Gly Gln Arg 150 155 160 GAA TTA ATT ATC GGA GAC CGT CAA ACT GGT AAA ACT TCA GTC GCT ATT 1281 Glu Leu Ile Ile Gly Asp Arg Gln Thr Gly Lys Thr Ser Val Ala Ile 165 170 175 180 GAT GCA ATC TTG AAC CAA AAA GGT CAA GAT ATG ATT TGT ATC TAT GTT 1329 Asp Ala Ile Leu Asn Gln Lys Gly Gln Asp Met Ile Cys Ile Tyr Val 185 190 195 GCG ATT GGA CAA AAA GAG TCA ACA GTT CGT ACA CAA GTT GAA ACG CTC 1377 Ala Ile Gly Gln Lys Glu Ser Thr Val Arg Thr Gln Val Glu Thr Leu 200 205 210 CGT AAA CTC GGT GCG ATG GAT TAT ACA ATC GTC GTA ACT GCG TCA GCT 1425 Arg Lys Leu Gly Ala Met Asp Tyr Thr Ile Val Val Thr Ala Ser Ala 215 220 225 TCT CAA CCT TCT CCA CTC CTT TAC ATC GCT CCT TAC GCT GGA GCT GCA 1473 Ser Gln Pro Ser Pro Leu Leu Tyr Ile Ala Pro Tyr Ala Gly Ala Ala 230 235 240 ATG GGT GAA GAA TTT ATG TAT AAC GGT AAA CAT GTC TTG GTT GTT TAT 1521 Met Gly Glu Glu Phe Met Tyr Asn Gly Lys His Val Leu Val Val Tyr 245 250 255 260 GAT GAT TTA TCT AAA CAA GCG GTC GCT TAC CGT GAA CTT TCT CTC TTG 1569 Asp Asp Leu Ser Lys Gln Ala Val Ala Tyr Arg Glu Leu Ser Leu Leu 265 270 275 CTC CGT CGT CCA CCA GGT CGT GAA GCA TAC CCA GGT GAC GTT TTC TAC 1617 Leu Arg Arg Pro Pro Gly Arg Glu Ala Tyr Pro Gly Asp Val Phe Tyr 280 285 290 TTG CAC TCA CGT CTT TTG GAA CGT GCT GCT AAA TTG TCT GAT GAT CTT 1665 Leu His Ser Arg Leu Leu Glu Arg Ala Ala Lys Leu Ser Asp Asp Leu 295 300 305 GGT GGT GGA TCA ATG ACG GCT TTG CCA TTC ATT GAA ACA CAA GCA GGT 1713 Gly Gly Gly Ser Met Thr Ala Leu Pro Phe Ile Glu Thr Gln Ala Gly 310 315 320 GAT ATC TCA GCT TAT ATT CCA ACA AAC GTT ATC TCT ATT ACC GAC GGT 1761 Asp Ile Ser Ala Tyr Ile Pro Thr Asn Val Ile Ser Ile Thr Asp Gly 325 330 335 340 CAA ATT TTC CTT GAA AAT GAC TTG TTC TAT TCA GGT GTA CGT CCT GCC 1809 Gln Ile Phe Leu Glu Asn Asp Leu Phe Tyr Ser Gly Val Arg Pro Ala 345 350 355 ATT GAT GCT GGT TCA TCA GTA TCA CGT GTT GGT GGT GCC GCA CAA ATC 1857 Ile Asp Ala Gly Ser Ser Val Ser Arg Val Gly Gly Ala Ala Gln Ile 360 365 370 AAA GCC ATG AAG AAA GTA GCT GGT ACT TTG CGT CTT GAC CTT GCG TCG 1905 Lys Ala Met Lys Lys Val Ala Gly Thr Leu Arg Leu Asp Leu Ala Ser 375 380 385 TTC CGT GAA CTT GAA GCC TTT ACA CAA TTT GGT TCT GAC CTT GAT GAA 1953 Phe Arg Glu Leu Glu Ala Phe Thr Gln Phe Gly Ser Asp Leu Asp Glu 390 395 400 GCG ACT CAA GCA AAA TTG AAT CGT GGT CGT CGT ACC GTT GAA GTC TTG 2001 Ala Thr Gln Ala Lys Leu Asn Arg Gly Arg Arg Thr Val Glu Val Leu 405 410 415 420 AAA CAA CCA TTG CAC AAA CCA TTG GCT GTT GAA AAA CAA GTT TTG ATT 2049 Lys Gln Pro Leu His Lys Pro Leu Ala Val Glu Lys Gln Val Leu Ile 425 430 435 CTC TAT GCA TTG ACT CAT GGT CAT CTT GAT AAT GTT CCA GTT GAT GAT 2097 Leu Tyr Ala Leu Thr His Gly His Leu Asp Asn Val Pro Val Asp Asp 440 445 450 GTT CTT GAT TTT GAA ACT AAA ATG TTC GAT TTC TTC GAT GCA AAT TAT 2145 Val Leu Asp Phe Glu Thr Lys Met Phe Asp Phe Phe Asp Ala Asn Tyr 455 460 465 GCA GAT CTC TTG AAC GTA ATT ACT GAC ACT AAA GAT TTG CCA GAA GAA 2193 Ala Asp Leu Leu Asn Val Ile Thr Asp Thr Lys Asp Leu Pro Glu Glu 470 475 480 GCA AAA CTT GAC GAA GCA ATT AAA GCA TTC AAA AAT ACA ACG AAT TAT 2241 Ala Lys Leu Asp Glu Ala Ile Lys Ala Phe Lys Asn Thr Thr Asn Tyr 485 490 495 500 TAATAAGGAG GCTAACTA ATG GGA GCT TCA CTT AAC GAA ATA AAA ACT AAG 2292 Met Gly Ala Ser Leu Asn Glu Ile Lys Thr Lys 1 5 10 ATT GCG TCA ACA AAG AAA ACA AGT CAA ATC ACA GGT GCC ATG CAA ATG 2340 Ile Ala Ser Thr Lys Lys Thr Ser Gln Ile Thr Gly Ala Met Gln Met 15 20 25 GTT TCT GCT GCT AAA CTT CAA AAA GCA GAA TCT CAC GCT AAA GCT TTT 2388 Val Ser Ala Ala Lys Leu Gln Lys Ala Glu Ser His Ala Lys Ala Phe 30 35 40 CAG ACT TAT GCT GAA AAA GTA CGT AAG ATT ACG ACT GAC TTA GTT TCA 2436 Gln Thr Tyr Ala Glu Lys Val Arg Lys Ile Thr Thr Asp Leu Val Ser 45 50 55 AGC GAT AAT GAG CCG GCC AAA AAT CCG ATG ATG ATT AAA CGT GAA GTC 2484 Ser Asp Asn Glu Pro Ala Lys Asn Pro Met Met Ile Lys Arg Glu Val 60 65 70 75 AAG AAA ACT GGC TAT CTC GTT ATC ACA TCA GAT CGT GGG CTT GTT GGC 2532 Lys Lys Thr Gly Tyr Leu Val Ile Thr Ser Asp Arg Gly Leu Val Gly 80 85 90 AGT TAT AAT TCA AAT ATT TTG AAG TCT GTT ATA AGT AAT ATA CGT AAA 2580 Ser Tyr Asn Ser Asn Ile Leu Lys Ser Val Ile Ser Asn Ile Arg Lys 95 100 105 CGC CAC ACA AAT GAG AGT GAG TAT ACA ATA CTT GCC CTT GGT GGT ACG 2628 Arg His Thr Asn Glu Ser Glu Tyr Thr Ile Leu Ala Leu Gly Gly Thr 110 115 120 GGA GCG GAC TTT TTC AAA GCC CGT AAC GTC AAA GTT TCT TAT GTT CTT 2676 Gly Ala Asp Phe Phe Lys Ala Arg Asn Val Lys Val Ser Tyr Val Leu 125 130 135 CGC GGA CTT TCA GAT CAA CCG ACC TTT GAA GAG GTT CGG GCA ATT GTT 2724 Arg Gly Leu Ser Asp Gln Pro Thr Phe Glu Glu Val Arg Ala Ile Val 140 145 150 155 ACA GAA GCC GTA GAA GAA TAT CAA GCA GAA GAA TTC GAT GAA CTC TAT 2772 Thr Glu Ala Val Glu Glu Tyr Gln Ala Glu Glu Phe Asp Glu Leu Tyr 160 165 170 GTT TGT TAC AAC CAC CAT GTG AAC TCA TTG GTA AGT GAG GCA CGG ATG 2820 Val Cys Tyr Asn His His Val Asn Ser Leu Val Ser Glu Ala Arg Met 175 180 185 GAA AAA ATG TTA CCT ATT TCT TTT GAT GAA AAA GGT GAC GAA AAA GCA 2868 Glu Lys Met Leu Pro Ile Ser Phe Asp Glu Lys Gly Asp Glu Lys Ala 190 195 200 TCT CTT GTT ACA TTT GAA TTA GAA CCA GAT CGT GAA ACA ATC TTA AAT 2916 Ser Leu Val Thr Phe Glu Leu Glu Pro Asp Arg Glu Thr Ile Leu Asn 205 210 215 CAG TTG TTG CCG CAA TAT GCT GAA AGT ATG ATT TAT GGC TCA ATT GTT 2964 Gln Leu Leu Pro Gln Tyr Ala Glu Ser Met Ile Tyr Gly Ser Ile Val 220 225 230 235 GAT GCA AAA ACA GCA GAA CAT GCT GCA GGT ATG ACC GCA ATG CGT ACT 3012 Asp Ala Lys Thr Ala Glu His Ala Ala Gly Met Thr Ala Met Arg Thr 240 245 250 GCA ACA GAT AAT GCA CAT TCT GTC ATT AAT GAT TTA ACC ATT CAA TAT 3060 Ala Thr Asp Asn Ala His Ser Val Ile Asn Asp Leu Thr Ile Gln Tyr 255 260 265 AAC CGT GCT CGT CAA GCT TCA ATT ACG CAA GAA ATT ACG GAA ATT GTT 3108 Asn Arg Ala Arg Gln Ala Ser Ile Thr Gln Glu Ile Thr Glu Ile Val 270 275 280 GCG GGT GCT TCA GCG CTA TAATTACTGT CAAACATTAT TCTCAATGTT 3156 Ala Gly Ala Ser Ala Leu 285 ACGATTTATC AACTTGAGGA ATAAATGTTC TGTCAGTAAA GGCTTTGAAT TTTAAATACG 3216 TTTGTCAGTA AATTTTTACT GATTAGCTTA AAAATGAATA GAAATTCTGT TGTTAGACAG 3276 AAAATAAAAA CAGGAGGAAA AACA TTG AGT TCT GGT AAA ATT ACT CAG GTT 3327 Leu Ser Ser Gly Lys Ile Thr Gln Val 1 5 ATC GGT CCC GTC GTT GAC GTG GAA TTT GGT TCT GAT GCC AAA CTG CCT 3375 Ile Gly Pro Val Val Asp Val Glu Phe Gly Ser Asp Ala Lys Leu Pro 10 15 20 25 GAG ATT AAC AAT GCC TTG ATT GTC TAC AAA GAT GTC AAT GGT TTA AAA 3423 Glu Ile Asn Asn Ala Leu Ile Val Tyr Lys Asp Val Asn Gly Leu Lys 30 35 40 ACA AAA ATT ACT CTT GAA GTT GCT TTG GAA CTT GGT GAT GGT GCA GTT 3471 Thr Lys Ile Thr Leu Glu Val Ala Leu Glu Leu Gly Asp Gly Ala Val 45 50 55 CGT ACG ATC GCT ATG GAA TCT ACT GAT GGA TTG ACT CGT GGA CTT GAA 3519 Arg Thr Ile Ala Met Glu Ser Thr Asp Gly Leu Thr Arg Gly Leu Glu 60 65 70 GTC CTT GAT ACA GGT AAA GCG GTC AGC GTT CCT GTT GGT GAA TCT ACT 3567 Val Leu Asp Thr Gly Lys Ala Val Ser Val Pro Val Gly Glu Ser Thr 75 80 85 CTT GGT CGT GTT TTT AAT GTC CTT GGT GAC GTT ATT GAT GGT GGA GAA 3615 Leu Gly Arg Val Phe Asn Val Leu Gly Asp Val Ile Asp Gly Gly Glu 90 95 100 105 GAT TTC CCT GCT GAT GCA GAA CGT AAT CCT ATC CAC AAG AAA GCT CCA 3663 Asp Phe Pro Ala Asp Ala Glu Arg Asn Pro Ile His Lys Lys Ala Pro 110 115 120 ACT TTT GAC GAA TTG TCA ACT GCA AAT GAA GTT CTT GTA ACA GGG ATT 3711 Thr Phe Asp Glu Leu Ser Thr Ala Asn Glu Val Leu Val Thr Gly Ile 125 130 135 AAA GTT GTC GAT TTA CTT GCC CCT TAT CTT AAA GGT GGG AAA GTC GGA 3759 Lys Val Val Asp Leu Leu Ala Pro Tyr Leu Lys Gly Gly Lys Val Gly 140 145 150 CTC TTC GGT GGT GCC GGT GTT GGT AAA ACC GTC CTT ATC CAA GAA TTG 3807 Leu Phe Gly Gly Ala Gly Val Gly Lys Thr Val Leu Ile Gln Glu Leu 155 160 165 ATT CAC AAT ATT GCC CAA GAA CAC GGT GGT ATT TCT GTA TTT ACA GGT 3855 Ile His Asn Ile Ala Gln Glu His Gly Gly Ile Ser Val Phe Thr Gly 170 175 180 185 GTT GGC GAT CGT ACT CGT GAC GGG AAT GAC CTT TAC TGG GAA ATG AAA 3903 Val Gly Asp Arg Thr Arg Asp Gly Asn Asp Leu Tyr Trp Glu Met Lys 190 195 200 GAA TCA GGC GTT ATT GAA AAA ACA GCC ATG GTC TTT GGT CAA ATG AAT 3951 Glu Ser Gly Val Ile Glu Lys Thr Ala Met Val Phe Gly Gln Met Asn 205 210 215 GAA CCA CCT GGA GCA CGT ATG CGT GTT GCC CTT ACT GGT TTA ACA ATT 3999 Glu Pro Pro Gly Ala Arg Met Arg Val Ala Leu Thr Gly Leu Thr Ile 220 225 230 GCG GAA TAT TTC CGT GAT GTT CAA GGA CAA GAC GTA TTG CTT TTC ATC 4047 Ala Glu Tyr Phe Arg Asp Val Gln Gly Gln Asp Val Leu Leu Phe Ile 235 240 245 GAT AAC ATC TTC CGT TTC ACT CAA GCT GGT TCA GAA GTT TCT GCC CTT 4095 Asp Asn Ile Phe Arg Phe Thr Gln Ala Gly Ser Glu Val Ser Ala Leu 250 255 260 265 TGG GGA CGT ATG CCT TCT GCC GTT GGT TAC CAA CCA ACT CTT GCA ACT 4143 Trp Gly Arg Met Pro Ser Ala Val Gly Tyr Gln Pro Thr Leu Ala Thr 270 275 280 GAA ATG GTT CAA TTA CAG GAA CGT ATC ACT TCT ACT AAG AAG GGT TCT 4191 Glu Met Val Gln Leu Gln Glu Arg Ile Thr Ser Thr Lys Lys Gly Ser 285 290 295 GTT ACA TCT ATC CCA GCG ATT TAT GTC CCT GCC GAT GAC TAT ACT GAC 4239 Val Thr Ser Ile Pro Ala Ile Tyr Val Pro Ala Asp Asp Tyr Thr Asp 300 305 310 CCA GCG CCA GCT ACA GCC TTC GCT CAC TTG GAC GCA ACA ACT AAC TTG 4287 Pro Ala Pro Ala Thr Ala Phe Ala His Leu Asp Ala Thr Thr Asn Leu 315 320 325 GAA CGT CGT TTG ACA CAA ATG GGT ATC TAT CCA GCC GTT GAC CCA CTT 4335 Glu Arg Arg Leu Thr Gln Met Gly Ile Tyr Pro Ala Val Asp Pro Leu 330 335 340 345 GCT TCA TCA TCA CGT GCG CTT ACA CCT GAA ATT GTT GGT GAA GAA CAC 4383 Ala Ser Ser Ser Arg Ala Leu Thr Pro Glu Ile Val Gly Glu Glu His 350 355 360 TAT GAA GTT GCA ATG GAA GTT CAA CGT GTC CTT CAA CGC TAC AAA GAA 4431 Tyr Glu Val Ala Met Glu Val Gln Arg Val Leu Gln Arg Tyr Lys Glu 365 370 375 TTG CAA GAT ATC ATT GCC ATT CTT GGT ATG GAT GAA TTG TCA GAT GAT 4479 Leu Gln Asp Ile Ile Ala Ile Leu Gly Met Asp Glu Leu Ser Asp Asp 380 385 390 GAA AAA ATT CTC GTT GGA CGT GCA CGT CGT ATC CAA TTC TTC CTT TCA 4527 Glu Lys Ile Leu Val Gly Arg Ala Arg Arg Ile Gln Phe Phe Leu Ser 395 400 405 CAA AAC TTC CAC GTT GCT GAA CAG TTT ACT GGT CAA CCT GGT TCA TAT 4575 Gln Asn Phe His Val Ala Glu Gln Phe Thr Gly Gln Pro Gly Ser Tyr 410 415 420 425 GTA CCA ATT GAC AAA ACA GTT CAT GAC TTC AAG GAA ATT TTG GAA GGT 4623 Val Pro Ile Asp Lys Thr Val His Asp Phe Lys Glu Ile Leu Glu Gly 430 435 440 AAA TAT GAC GAA GTC CCT GAA GAT GCT TTC CGT GGA GTA GGT CCA ATT 4671 Lys Tyr Asp Glu Val Pro Glu Asp Ala Phe Arg Gly Val Gly Pro Ile 445 450 455 GAA GAC GTA CTT GCA AAA GCA AAA TCA ATG GGT TAT TAATTCGATT 4717 Glu Asp Val Leu Ala Lys Ala Lys Ser Met Gly Tyr 460 465 TCTTATGAAA TGACAAAGTG AAAATACATT ATTGAATCGC AAAATTTACT GACAATAATT 4777 CTGTCGTAAG TGCTCACTTT TAAGTTGTTC CGATCGTT 4815 175 amino acids amino acid linear protein 2 Met Thr Lys Val Asn Ser Gln Lys Tyr Ser Lys Ala Leu Leu Glu Val 1 5 10 15 Ala Arg Glu Lys Gly Gln Leu Glu Ala Ile Leu Thr Glu Val Ser Glu 20 25 30 Met Ile Gln Leu Phe Lys Glu Asn Asn Leu Gly Ala Phe Leu Ala Asn 35 40 45 Glu Val Tyr Ser Phe Ser Ala Lys Ser Glu Leu Ile Asp Thr Leu Leu 50 55 60 Gln Thr Ser Ser Glu Val Met Ser Asn Phe Leu Asn Thr Ile Arg Ser 65 70 75 80 Asn Gly Arg Leu Ala Asp Leu Gly Glu Ile Leu Glu Glu Thr Lys Asn 85 90 95 Ala Ala Asp Asp Met Phe Lys Ile Ala Asp Val Glu Val Val Ser Ser 100 105 110 Ile Ala Leu Ser Glu Ala Gln Ile Glu Lys Phe Lys Ala Met Ala Lys 115 120 125 Ser Lys Phe Asp Leu Asn Glu Val Thr Val Ile Asn Thr Val Asn Glu 130 135 140 Lys Ile Leu Gly Gly Phe Ile Val Asn Ser Arg Gly Lys Ile Ile Asp 145 150 155 160 Ala Ser Leu Lys Thr Gln Leu Ala Lys Ile Ala Ala Glu Ile Leu 165 170 175 500 amino acids amino acid linear protein 3 Leu Ala Ile Lys Ala Asn Glu Ile Ser Ser Leu Ile Lys Lys Gln Ile 1 5 10 15 Glu Asn Phe Thr Pro Asp Phe Glu Val Ala Glu Thr Gly Val Val Thr 20 25 30 Tyr Val Gly Asp Gly Ile Ala Arg Ala Tyr Gly Leu Glu Asn Ala Met 35 40 45 Ser Gly Glu Leu Val Glu Phe Ser Asn Gly Ile Leu Gly Met Ala Gln 50 55 60 Asn Leu Asp Ala Thr Asp Val Gly Ile Ile Val Leu Gly Asp Phe Leu 65 70 75 80 Ser Ile Arg Glu Gly Asp Thr Val Lys Arg Thr Gly Lys Ile Met Glu 85 90 95 Ile Gln Val Gly Glu Glu Leu Ile Gly Arg Val Val Asn Pro Leu Gly 100 105 110 Gln Pro Val Asp Gly Leu Gly Glu Leu Asn Thr Gly Lys Thr Arg Pro 115 120 125 Val Glu Ala Lys Ala Pro Gly Val Met Gln Arg Lys Ser Val Ser Glu 130 135 140 Pro Leu Gln Thr Gly Leu Lys Ala Ile Asp Ala Leu Val Pro Ile Gly 145 150 155 160 Arg Gly Gln Arg Glu Leu Ile Ile Gly Asp Arg Gln Thr Gly Lys Thr 165 170 175 Ser Val Ala Ile Asp Ala Ile Leu Asn Gln Lys Gly Gln Asp Met Ile 180 185 190 Cys Ile Tyr Val Ala Ile Gly Gln Lys Glu Ser Thr Val Arg Thr Gln 195 200 205 Val Glu Thr Leu Arg Lys Leu Gly Ala Met Asp Tyr Thr Ile Val Val 210 215 220 Thr Ala Ser Ala Ser Gln Pro Ser Pro Leu Leu Tyr Ile Ala Pro Tyr 225 230 235 240 Ala Gly Ala Ala Met Gly Glu Glu Phe Met Tyr Asn Gly Lys His Val 245 250 255 Leu Val Val Tyr Asp Asp Leu Ser Lys Gln Ala Val Ala Tyr Arg Glu 260 265 270 Leu Ser Leu Leu Leu Arg Arg Pro Pro Gly Arg Glu Ala Tyr Pro Gly 275 280 285 Asp Val Phe Tyr Leu His Ser Arg Leu Leu Glu Arg Ala Ala Lys Leu 290 295 300 Ser Asp Asp Leu Gly Gly Gly Ser Met Thr Ala Leu Pro Phe Ile Glu 305 310 315 320 Thr Gln Ala Gly Asp Ile Ser Ala Tyr Ile Pro Thr Asn Val Ile Ser 325 330 335 Ile Thr Asp Gly Gln Ile Phe Leu Glu Asn Asp Leu Phe Tyr Ser Gly 340 345 350 Val Arg Pro Ala Ile Asp Ala Gly Ser Ser Val Ser Arg Val Gly Gly 355 360 365 Ala Ala Gln Ile Lys Ala Met Lys Lys Val Ala Gly Thr Leu Arg Leu 370 375 380 Asp Leu Ala Ser Phe Arg Glu Leu Glu Ala Phe Thr Gln Phe Gly Ser 385 390 395 400 Asp Leu Asp Glu Ala Thr Gln Ala Lys Leu Asn Arg Gly Arg Arg Thr 405 410 415 Val Glu Val Leu Lys Gln Pro Leu His Lys Pro Leu Ala Val Glu Lys 420 425 430 Gln Val Leu Ile Leu Tyr Ala Leu Thr His Gly His Leu Asp Asn Val 435 440 445 Pro Val Asp Asp Val Leu Asp Phe Glu Thr Lys Met Phe Asp Phe Phe 450 455 460 Asp Ala Asn Tyr Ala Asp Leu Leu Asn Val Ile Thr Asp Thr Lys Asp 465 470 475 480 Leu Pro Glu Glu Ala Lys Leu Asp Glu Ala Ile Lys Ala Phe Lys Asn 485 490 495 Thr Thr Asn Tyr 500 289 amino acids amino acid linear protein 4 Met Gly Ala Ser Leu Asn Glu Ile Lys Thr Lys Ile Ala Ser Thr Lys 1 5 10 15 Lys Thr Ser Gln Ile Thr Gly Ala Met Gln Met Val Ser Ala Ala Lys 20 25 30 Leu Gln Lys Ala Glu Ser His Ala Lys Ala Phe Gln Thr Tyr Ala Glu 35 40 45 Lys Val Arg Lys Ile Thr Thr Asp Leu Val Ser Ser Asp Asn Glu Pro 50 55 60 Ala Lys Asn Pro Met Met Ile Lys Arg Glu Val Lys Lys Thr Gly Tyr 65 70 75 80 Leu Val Ile Thr Ser Asp Arg Gly Leu Val Gly Ser Tyr Asn Ser Asn 85 90 95 Ile Leu Lys Ser Val Ile Ser Asn Ile Arg Lys Arg His Thr Asn Glu 100 105 110 Ser Glu Tyr Thr Ile Leu Ala Leu Gly Gly Thr Gly Ala Asp Phe Phe 115 120 125 Lys Ala Arg Asn Val Lys Val Ser Tyr Val Leu Arg Gly Leu Ser Asp 130 135 140 Gln Pro Thr Phe Glu Glu Val Arg Ala Ile Val Thr Glu Ala Val Glu 145 150 155 160 Glu Tyr Gln Ala Glu Glu Phe Asp Glu Leu Tyr Val Cys Tyr Asn His 165 170 175 His Val Asn Ser Leu Val Ser Glu Ala Arg Met Glu Lys Met Leu Pro 180 185 190 Ile Ser Phe Asp Glu Lys Gly Asp Glu Lys Ala Ser Leu Val Thr Phe 195 200 205 Glu Leu Glu Pro Asp Arg Glu Thr Ile Leu Asn Gln Leu Leu Pro Gln 210 215 220 Tyr Ala Glu Ser Met Ile Tyr Gly Ser Ile Val Asp Ala Lys Thr Ala 225 230 235 240 Glu His Ala Ala Gly Met Thr Ala Met Arg Thr Ala Thr Asp Asn Ala 245 250 255 His Ser Val Ile Asn Asp Leu Thr Ile Gln Tyr Asn Arg Ala Arg Gln 260 265 270 Ala Ser Ile Thr Gln Glu Ile Thr Glu Ile Val Ala Gly Ala Ser Ala 275 280 285 Leu 469 amino acids amino acid linear protein 5 Leu Ser Ser Gly Lys Ile Thr Gln Val Ile Gly Pro Val Val Asp Val 1 5 10 15 Glu Phe Gly Ser Asp Ala Lys Leu Pro Glu Ile Asn Asn Ala Leu Ile 20 25 30 Val Tyr Lys Asp Val Asn Gly Leu Lys Thr Lys Ile Thr Leu Glu Val 35 40 45 Ala Leu Glu Leu Gly Asp Gly Ala Val Arg Thr Ile Ala Met Glu Ser 50 55 60 Thr Asp Gly Leu Thr Arg Gly Leu Glu Val Leu Asp Thr Gly Lys Ala 65 70 75 80 Val Ser Val Pro Val Gly Glu Ser Thr Leu Gly Arg Val Phe Asn Val 85 90 95 Leu Gly Asp Val Ile Asp Gly Gly Glu Asp Phe Pro Ala Asp Ala Glu 100 105 110 Arg Asn Pro Ile His Lys Lys Ala Pro Thr Phe Asp Glu Leu Ser Thr 115 120 125 Ala Asn Glu Val Leu Val Thr Gly Ile Lys Val Val Asp Leu Leu Ala 130 135 140 Pro Tyr Leu Lys Gly Gly Lys Val Gly Leu Phe Gly Gly Ala Gly Val 145 150 155 160 Gly Lys Thr Val Leu Ile Gln Glu Leu Ile His Asn Ile Ala Gln Glu 165 170 175 His Gly Gly Ile Ser Val Phe Thr Gly Val Gly Asp Arg Thr Arg Asp 180 185 190 Gly Asn Asp Leu Tyr Trp Glu Met Lys Glu Ser Gly Val Ile Glu Lys 195 200 205 Thr Ala Met Val Phe Gly Gln Met Asn Glu Pro Pro Gly Ala Arg Met 210 215 220 Arg Val Ala Leu Thr Gly Leu Thr Ile Ala Glu Tyr Phe Arg Asp Val 225 230 235 240 Gln Gly Gln Asp Val Leu Leu Phe Ile Asp Asn Ile Phe Arg Phe Thr 245 250 255 Gln Ala Gly Ser Glu Val Ser Ala Leu Trp Gly Arg Met Pro Ser Ala 260 265 270 Val Gly Tyr Gln Pro Thr Leu Ala Thr Glu Met Val Gln Leu Gln Glu 275 280 285 Arg Ile Thr Ser Thr Lys Lys Gly Ser Val Thr Ser Ile Pro Ala Ile 290 295 300 Tyr Val Pro Ala Asp Asp Tyr Thr Asp Pro Ala Pro Ala Thr Ala Phe 305 310 315 320 Ala His Leu Asp Ala Thr Thr Asn Leu Glu Arg Arg Leu Thr Gln Met 325 330 335 Gly Ile Tyr Pro Ala Val Asp Pro Leu Ala Ser Ser Ser Arg Ala Leu 340 345 350 Thr Pro Glu Ile Val Gly Glu Glu His Tyr Glu Val Ala Met Glu Val 355 360 365 Gln Arg Val Leu Gln Arg Tyr Lys Glu Leu Gln Asp Ile Ile Ala Ile 370 375 380 Leu Gly Met Asp Glu Leu Ser Asp Asp Glu Lys Ile Leu Val Gly Arg 385 390 395 400 Ala Arg Arg Ile Gln Phe Phe Leu Ser Gln Asn Phe His Val Ala Glu 405 410 415 Gln Phe Thr Gly Gln Pro Gly Ser Tyr Val Pro Ile Asp Lys Thr Val 420 425 430 His Asp Phe Lys Glu Ile Leu Glu Gly Lys Tyr Asp Glu Val Pro Glu 435 440 445 Asp Ala Phe Arg Gly Val Gly Pro Ile Glu Asp Val Leu Ala Lys Ala 450 455 460 Lys Ser Met Gly Tyr 465 2207 base pairs nucleic acid double linear DNA (genomic) NO NO Lactococcus lactis subsp. lactis CDS 4..633 /partial /codon_start= 4 /product= “ATPase subunit, partial sequence” /gene= “atpA” /standard_name= “alpha subunit of the F1 portion of the F0F1 ATPase” /label= alpha-subunit CDS 652..1518 /codon_start= 652 /product= “ATPase subunit” /gene= “atpG” /standard_name= “gamma subunit of the F1 portion of the F0F1 ATPase” /label= gamma-subunit CDS 1654..2205 /partial /codon_start= 1654 /product= “ATPase subunit, partial sequence” /gene= “atpD” /standard_name= “beta subunit of the F1 portion of the F0F1 ATPase” /label= beta-subunit 6 TGA TTC TAC TTA CAT TCA CGT CTT TTG GAA CGT GCT GCC AAA TTA TCT 48 Phe Tyr Leu His Ser Arg Leu Leu Glu Arg Ala Ala Lys Leu Ser 470 475 480 GAC TAT CTT GGT GGT GGT TCA ATG ACT GCA CTG CCA TTC ATT GAA ACA 96 Asp Tyr Leu Gly Gly Gly Ser Met Thr Ala Leu Pro Phe Ile Glu Thr 485 490 495 500 CAA GCC GGA GAT ATC TCA GCT TAT ATT GCA ACA AAC GTT ATC TCT ATT 144 Gln Ala Gly Asp Ile Ser Ala Tyr Ile Ala Thr Asn Val Ile Ser Ile 505 510 515 ACT GAC GGT CAA ATT TTC CTT GAA AAT GAC TTA TTC TAT TCA GGT GTA 192 Thr Asp Gly Gln Ile Phe Leu Glu Asn Asp Leu Phe Tyr Ser Gly Val 520 525 530 CGT CCT GCC ATC GAT GCT GGT TCT TCA GTT TCT CGG GTT GGT GGT GCT 240 Arg Pro Ala Ile Asp Ala Gly Ser Ser Val Ser Arg Val Gly Gly Ala 535 540 545 GCA CAG ATC AAA GCC ATG AAG AAA GTT GCT GGT ACT TTG CGT CTT GAC 288 Ala Gln Ile Lys Ala Met Lys Lys Val Ala Gly Thr Leu Arg Leu Asp 550 555 560 CTT GCG TCA TTC CGT GAA CTT GAA GCC TTT ACT CAA TTT GGT TCT GAT 336 Leu Ala Ser Phe Arg Glu Leu Glu Ala Phe Thr Gln Phe Gly Ser Asp 565 570 575 580 CTT GAT GAA GCG ACT CAA GCA AAA TTG AAT CGT GGT CGT CGT ACC GTT 384 Leu Asp Glu Ala Thr Gln Ala Lys Leu Asn Arg Gly Arg Arg Thr Val 585 590 595 GAA GTT TTG AAG CAA CCA TTG CAC AAA CCA TTG GCT GTT GAA AAA CAA 432 Glu Val Leu Lys Gln Pro Leu His Lys Pro Leu Ala Val Glu Lys Gln 600 605 610 GTT TTA ATT CTT TAT GCA TTG ACT CAT GGT CAC TTG GAT GAT GTT CCA 480 Val Leu Ile Leu Tyr Ala Leu Thr His Gly His Leu Asp Asp Val Pro 615 620 625 GTT GAT GAC GTC CTT GAT TTT GAA ACA AAC AAT GTC CGA TTC TTC GAT 528 Val Asp Asp Val Leu Asp Phe Glu Thr Asn Asn Val Arg Phe Phe Asp 630 635 640 GCA AAT TAT GCA AAA CTC TTG AAC GTG ATT ACT GAA ACT AAA GAT TGC 576 Ala Asn Tyr Ala Lys Leu Leu Asn Val Ile Thr Glu Thr Lys Asp Cys 645 650 655 660 CAG AAG AAG CAA AAC TCG ACG AAG CAA TTA AAG CAT TCT AAA ATA CAA 624 Gln Lys Lys Gln Asn Ser Thr Lys Gln Leu Lys His Ser Lys Ile Gln 665 670 675 CGA ATT ATT AATAAGGAGG CTAATCTA ATG GGA GCT TCA CTT AAT GAA ATA 675 Arg Ile Ile Met Gly Ala Ser Leu Asn Glu Ile 1 5 AAA ACT AAG ATT GCC TCA ACG AAG AAA ACA AGT CAA ATA ACT GGA GCC 723 Lys Thr Lys Ile Ala Ser Thr Lys Lys Thr Ser Gln Ile Thr Gly Ala 10 15 20 ATG CAA ATG GTT TCC GCT GCG AAA CTT CAA AAA GCT GAA TCT CAT GCC 771 Met Gln Met Val Ser Ala Ala Lys Leu Gln Lys Ala Glu Ser His Ala 25 30 35 40 AAA GCA TTT CAA ATT TAT GCT GAA AAA GTT CGT AAA ATT ACA ACT GAT 819 Lys Ala Phe Gln Ile Tyr Ala Glu Lys Val Arg Lys Ile Thr Thr Asp 45 50 55 TTA GTT TCC TCT GAC AAA GAG CCA GCT AAG AAT CCA ATG ATG ATA GGA 867 Leu Val Ser Ser Asp Lys Glu Pro Ala Lys Asn Pro Met Met Ile Gly 60 65 70 AGA GAA GTC AAA AAA ACT GGC TAT CTT GTA ATT ACT TCG GAT CGT GGA 915 Arg Glu Val Lys Lys Thr Gly Tyr Leu Val Ile Thr Ser Asp Arg Gly 75 80 85 CTT GTC GGT GGC TAT AAT TCA TAT ATT TTG AAA TCT GTC ATG AAT ACT 963 Leu Val Gly Gly Tyr Asn Ser Tyr Ile Leu Lys Ser Val Met Asn Thr 90 95 100 ATC CGT AAA CGT CCT GCT AAT GAA AGT GAA TAT ACT ATT CTT GCA CTT 1011 Ile Arg Lys Arg Pro Ala Asn Glu Ser Glu Tyr Thr Ile Leu Ala Leu 105 110 115 120 GGC GGT ACT GGA GCA GAT TTC TTC GGA GCA AGC AAT GTT AAA AGT TTC 1059 Gly Gly Thr Gly Ala Asp Phe Phe Gly Ala Ser Asn Val Lys Ser Phe 125 130 135 TTA GTC CTT TGT GGT TTT TCA GAC CAA CCA AAT TTT GAA GAA GTT AGA 1107 Leu Val Leu Cys Gly Phe Ser Asp Gln Pro Asn Phe Glu Glu Val Arg 140 145 150 GCG ATT GTT ACA GAA GCG GTA ACT GAA TAT CAA GCA GAA GAA TTT GAT 1155 Ala Ile Val Thr Glu Ala Val Thr Glu Tyr Gln Ala Glu Glu Phe Asp 155 160 165 GAA CTT TAT GTT TGC TAT AAT CAC CAT GTG AAC TCA TTG GTA AGT GAA 1203 Glu Leu Tyr Val Cys Tyr Asn His His Val Asn Ser Leu Val Ser Glu 170 175 180 GCA AGT ATG GAA AAA ATG TTG CCT ATT TTT TTT GAA GCA TCA GGT CAA 1251 Ala Ser Met Glu Lys Met Leu Pro Ile Phe Phe Glu Ala Ser Gly Gln 185 190 195 200 CAA AAA CCA TTT TTT GAA ACA TTT GAA TTA GAA CCA GAT TGT GAA ACA 1299 Gln Lys Pro Phe Phe Glu Thr Phe Glu Leu Glu Pro Asp Cys Glu Thr 205 210 215 ATT TTA AAC CAA TTG TTG CCA CCA TAC GCT GAA AGT ATG ATT TAT GGT 1347 Ile Leu Asn Gln Leu Leu Pro Pro Tyr Ala Glu Ser Met Ile Tyr Gly 220 225 230 TCA ATC GTT GAT GCT AAG ACA GCA GAA CAT GCT GCA GGT ATG ACA GCA 1395 Ser Ile Val Asp Ala Lys Thr Ala Glu His Ala Ala Gly Met Thr Ala 235 240 245 ATG CGT ACT GCA ACT GAT AAT GCT CAC TCT GTT ATC AAT GAT TTG ACT 1443 Met Arg Thr Ala Thr Asp Asn Ala His Ser Val Ile Asn Asp Leu Thr 250 255 260 ATT CAA TAC AAC CGT GCT CGT CAA GCA TCG ATT ACG CAA GAA ATT ACG 1491 Ile Gln Tyr Asn Arg Ala Arg Gln Ala Ser Ile Thr Gln Glu Ile Thr 265 270 275 280 GAA ATC GTT GCA GGA GCC TCA GCG CTT TAATTTACTG ATAGGAATTC 1538 Glu Ile Val Ala Gly Ala Ser Ala Leu 285 TGTCAGTGAT GGCTTTGAAT CTTAATTGTT TTTGTCAGTA AAATTTTTAC TGACAAACAT 1598 AAAAATGAAT AGAAATTCTG TTCTTTGACA GAAAATAAAA ACAGGAGGAA AAACA TTG 1656 Leu 1 AGT TCT GGT AAA ATT ACT CAG ATT ATC GGT CCC GTC GTT GAC GTG GAA 1704 Ser Ser Gly Lys Ile Thr Gln Ile Ile Gly Pro Val Val Asp Val Glu 5 10 15 TTT GGT TCT GAT GCC AAA TTG CCT GAG ATT AAC AAT GCC TTG ATT GTC 1752 Phe Gly Ser Asp Ala Lys Leu Pro Glu Ile Asn Asn Ala Leu Ile Val 20 25 30 TAC AAA GAT GTC AAT GGC CTA AAA ACA AAA ATT ACT CTT GAA GTT GCT 1800 Tyr Lys Asp Val Asn Gly Leu Lys Thr Lys Ile Thr Leu Glu Val Ala 35 40 45 TTG GAA CTT GGT GAT GGT GCA GTT CGT ACA ATC GCT ATG GAA TCT ACT 1848 Leu Glu Leu Gly Asp Gly Ala Val Arg Thr Ile Ala Met Glu Ser Thr 50 55 60 65 GAT GGC TTG ACT CGT GGA CTT GAA GTC CTT GAT ACA GGT AAA GCA GTC 1896 Asp Gly Leu Thr Arg Gly Leu Glu Val Leu Asp Thr Gly Lys Ala Val 70 75 80 AGC GTT CCT GTT GGG GAA GCC ACT CTT GGT CGT GTT TTT AAC GTC CTT 1944 Ser Val Pro Val Gly Glu Ala Thr Leu Gly Arg Val Phe Asn Val Leu 85 90 95 GGT GAT GTT ATT GAC GGT GGG GAA GAA TTT GCT GCT GAT GCA GAA CGT 1992 Gly Asp Val Ile Asp Gly Gly Glu Glu Phe Ala Ala Asp Ala Glu Arg 100 105 110 AAT CCT ATC CAT AAA AAA GCT CCA ACA TTT GAC GAA TTG TCA ACT GCA 2040 Asn Pro Ile His Lys Lys Ala Pro Thr Phe Asp Glu Leu Ser Thr Ala 115 120 125 AAC GAA GTT CTC GTA ACT GGG ATT AAA GTT GTC GAT TTG CTT GCA CCT 2088 Asn Glu Val Leu Val Thr Gly Ile Lys Val Val Asp Leu Leu Ala Pro 130 135 140 145 TAC CTT AAA GGT GGT AAA GTT GGA CTT TTC GGT GGT GCC GGA GTT GGT 2136 Tyr Leu Lys Gly Gly Lys Val Gly Leu Phe Gly Gly Ala Gly Val Gly 150 155 160 AAA GCC GTC CTT ATT CAA GAA TTG AAA CAC AAC ATC GCC CAA GAA CAC 2184 Lys Ala Val Leu Ile Gln Glu Leu Lys His Asn Ile Ala Gln Glu His 165 170 175 GGA GGT ATT TCT GTG TTT ACC GG 2207 Gly Gly Ile Ser Val Phe Thr 180 210 amino acids amino acid linear protein 7 Phe Tyr Leu His Ser Arg Leu Leu Glu Arg Ala Ala Lys Leu Ser Asp 1 5 10 15 Tyr Leu Gly Gly Gly Ser Met Thr Ala Leu Pro Phe Ile Glu Thr Gln 20 25 30 Ala Gly Asp Ile Ser Ala Tyr Ile Ala Thr Asn Val Ile Ser Ile Thr 35 40 45 Asp Gly Gln Ile Phe Leu Glu Asn Asp Leu Phe Tyr Ser Gly Val Arg 50 55 60 Pro Ala Ile Asp Ala Gly Ser Ser Val Ser Arg Val Gly Gly Ala Ala 65 70 75 80 Gln Ile Lys Ala Met Lys Lys Val Ala Gly Thr Leu Arg Leu Asp Leu 85 90 95 Ala Ser Phe Arg Glu Leu Glu Ala Phe Thr Gln Phe Gly Ser Asp Leu 100 105 110 Asp Glu Ala Thr Gln Ala Lys Leu Asn Arg Gly Arg Arg Thr Val Glu 115 120 125 Val Leu Lys Gln Pro Leu His Lys Pro Leu Ala Val Glu Lys Gln Val 130 135 140 Leu Ile Leu Tyr Ala Leu Thr His Gly His Leu Asp Asp Val Pro Val 145 150 155 160 Asp Asp Val Leu Asp Phe Glu Thr Asn Asn Val Arg Phe Phe Asp Ala 165 170 175 Asn Tyr Ala Lys Leu Leu Asn Val Ile Thr Glu Thr Lys Asp Cys Gln 180 185 190 Lys Lys Gln Asn Ser Thr Lys Gln Leu Lys His Ser Lys Ile Gln Arg 195 200 205 Ile Ile 210 289 amino acids amino acid linear protein 8 Met Gly Ala Ser Leu Asn Glu Ile Lys Thr Lys Ile Ala Ser Thr Lys 1 5 10 15 Lys Thr Ser Gln Ile Thr Gly Ala Met Gln Met Val Ser Ala Ala Lys 20 25 30 Leu Gln Lys Ala Glu Ser His Ala Lys Ala Phe Gln Ile Tyr Ala Glu 35 40 45 Lys Val Arg Lys Ile Thr Thr Asp Leu Val Ser Ser Asp Lys Glu Pro 50 55 60 Ala Lys Asn Pro Met Met Ile Gly Arg Glu Val Lys Lys Thr Gly Tyr 65 70 75 80 Leu Val Ile Thr Ser Asp Arg Gly Leu Val Gly Gly Tyr Asn Ser Tyr 85 90 95 Ile Leu Lys Ser Val Met Asn Thr Ile Arg Lys Arg Pro Ala Asn Glu 100 105 110 Ser Glu Tyr Thr Ile Leu Ala Leu Gly Gly Thr Gly Ala Asp Phe Phe 115 120 125 Gly Ala Ser Asn Val Lys Ser Phe Leu Val Leu Cys Gly Phe Ser Asp 130 135 140 Gln Pro Asn Phe Glu Glu Val Arg Ala Ile Val Thr Glu Ala Val Thr 145 150 155 160 Glu Tyr Gln Ala Glu Glu Phe Asp Glu Leu Tyr Val Cys Tyr Asn His 165 170 175 His Val Asn Ser Leu Val Ser Glu Ala Ser Met Glu Lys Met Leu Pro 180 185 190 Ile Phe Phe Glu Ala Ser Gly Gln Gln Lys Pro Phe Phe Glu Thr Phe 195 200 205 Glu Leu Glu Pro Asp Cys Glu Thr Ile Leu Asn Gln Leu Leu Pro Pro 210 215 220 Tyr Ala Glu Ser Met Ile Tyr Gly Ser Ile Val Asp Ala Lys Thr Ala 225 230 235 240 Glu His Ala Ala Gly Met Thr Ala Met Arg Thr Ala Thr Asp Asn Ala 245 250 255 His Ser Val Ile Asn Asp Leu Thr Ile Gln Tyr Asn Arg Ala Arg Gln 260 265 270 Ala Ser Ile Thr Gln Glu Ile Thr Glu Ile Val Ala Gly Ala Ser Ala 275 280 285 Leu 184 amino acids amino acid linear protein 9 Leu Ser Ser Gly Lys Ile Thr Gln Ile Ile Gly Pro Val Val Asp Val 1 5 10 15 Glu Phe Gly Ser Asp Ala Lys Leu Pro Glu Ile Asn Asn Ala Leu Ile 20 25 30 Val Tyr Lys Asp Val Asn Gly Leu Lys Thr Lys Ile Thr Leu Glu Val 35 40 45 Ala Leu Glu Leu Gly Asp Gly Ala Val Arg Thr Ile Ala Met Glu Ser 50 55 60 Thr Asp Gly Leu Thr Arg Gly Leu Glu Val Leu Asp Thr Gly Lys Ala 65 70 75 80 Val Ser Val Pro Val Gly Glu Ala Thr Leu Gly Arg Val Phe Asn Val 85 90 95 Leu Gly Asp Val Ile Asp Gly Gly Glu Glu Phe Ala Ala Asp Ala Glu 100 105 110 Arg Asn Pro Ile His Lys Lys Ala Pro Thr Phe Asp Glu Leu Ser Thr 115 120 125 Ala Asn Glu Val Leu Val Thr Gly Ile Lys Val Val Asp Leu Leu Ala 130 135 140 Pro Tyr Leu Lys Gly Gly Lys Val Gly Leu Phe Gly Gly Ala Gly Val 145 150 155 160 Gly Lys Ala Val Leu Ile Gln Glu Leu Lys His Asn Ile Ala Gln Glu 165 170 175 His Gly Gly Ile Ser Val Phe Thr 180 2161 base pairs nucleic acid double linear DNA (genomic) NO NO Streptococcus thermophilus ST3 CDS 2..637 /partial /codon_start= 2 /product= “ATPase subunit, partial sequence” /gene= “atpA” /standard_name= “alpha subunit of the F1 portion of the F0F1 ATPase” /label= alpha-subunit CDS 659..1537 /codon_start= 659 /product= “ATPase subunit” /gene= “atpG” /standard_name= “gamma subunit of the F1 portion of the F0F1 ATPase” /label= gamma-subunit CDS 1616..2161 /partial /codon_start= 1616 /product= “ATPase subunit, partial sequence” /gene= “atpD” /standard_name= “beta subunit of the F1 portion of the F0F1 ATPase” /label= beta-subunit 10 T GAT TCT CAT CTC CAC TCA CGT CTT TTG GAA CGT TCA GCT AAG CTT 46 Asp Ser His Leu His Ser Arg Leu Leu Glu Arg Ser Ala Lys Leu 185 190 195 TCA GAT GAT CTT GGT GGT GGT TCA ATG ACT GCC TTG CCA ATC ATC CAA 94 Ser Asp Asp Leu Gly Gly Gly Ser Met Thr Ala Leu Pro Ile Ile Gln 200 205 210 215 ACA CAA GCA GGA GAT ATC TCA GCT TAT ATC GCG ACA AAC GTT ATT TCT 142 Thr Gln Ala Gly Asp Ile Ser Ala Tyr Ile Ala Thr Asn Val Ile Ser 220 225 230 ATC ACA GAT GGA CAA ATC TTC TTG CAA GAA AAT CTT TTC AAC TCA GGT 190 Ile Thr Asp Gly Gln Ile Phe Leu Gln Glu Asn Leu Phe Asn Ser Gly 235 240 245 ATT CGT CCT GCG ATT GAT GCT GGT TCT TCA GTA TCA CGT GTT GGT GGT 238 Ile Arg Pro Ala Ile Asp Ala Gly Ser Ser Val Ser Arg Val Gly Gly 250 255 260 TCA GCA CAA ATC AAA GCA ATG AAG AAA GTT GCT GGT ACC CTT CGT CTT 286 Ser Ala Gln Ile Lys Ala Met Lys Lys Val Ala Gly Thr Leu Arg Leu 265 270 275 GAC TTG GCT TCT CAC CGT GAA CTT GAA GCC TTT ACA CAA TTC GGT TCT 334 Asp Leu Ala Ser His Arg Glu Leu Glu Ala Phe Thr Gln Phe Gly Ser 280 285 290 295 GAT TTG GAT GCC GCA ACA CAA GCT AAA CTT AAT CGT GGA CGT CGT ACA 382 Asp Leu Asp Ala Ala Thr Gln Ala Lys Leu Asn Arg Gly Arg Arg Thr 300 305 310 GTT GAA GTG CTT AAA CAA CCA CTT CAT AAC CCA CTT CCG GTT GAA AAA 430 Val Glu Val Leu Lys Gln Pro Leu His Asn Pro Leu Pro Val Glu Lys 315 320 325 CAA GTT CTT ATT CTT TAC GCT TTG ACA CAT GGC TTC TTG GAC AGT GTT 478 Gln Val Leu Ile Leu Tyr Ala Leu Thr His Gly Phe Leu Asp Ser Val 330 335 340 CCG GTT GAT CAA ATC TTG GAT TTT GAA GAA GCC CTC TAT GAC TAC TTT 526 Pro Val Asp Gln Ile Leu Asp Phe Glu Glu Ala Leu Tyr Asp Tyr Phe 345 350 355 GAT AGC CAT CAT GAG GAT ATC TTT GAA ACA ATC CGT TCA ACT AAG GAT 574 Asp Ser His His Glu Asp Ile Phe Glu Thr Ile Arg Ser Thr Lys Asp 360 365 370 375 CTT CCT GAA GAA GCT GTG CTT AAT GAA GCT ATC CAA GCT TTC AAA GAT 622 Leu Pro Glu Glu Ala Val Leu Asn Glu Ala Ile Gln Ala Phe Lys Asp 380 385 390 CAA TCG GAA TAC AAA TAGAGATAGG GAGGACAGCA T ATG GCA GGC TCT CTA 673 Gln Ser Glu Tyr Lys Met Ala Gly Ser Leu 395 1 5 AGA GAA ATC AAA GCA AAA ATT GCT TCA ATT AAG CAA ACG AGT CAT ATT 721 Arg Glu Ile Lys Ala Lys Ile Ala Ser Ile Lys Gln Thr Ser His Ile 10 15 20 ACA GGA GCC ATG CAA ATG GTT TCT GCT TCT AAA TTG ACA CGT TCT GAG 769 Thr Gly Ala Met Gln Met Val Ser Ala Ser Lys Leu Thr Arg Ser Glu 25 30 35 CAG GCT GCT AAA GAT TTC CAA ATC TAT GCC TCA AAA ATT AGA CAG ATC 817 Gln Ala Ala Lys Asp Phe Gln Ile Tyr Ala Ser Lys Ile Arg Gln Ile 40 45 50 ACA ACA GAT CTT CTA CAT TCA GAA TTG GTT AAT GGT TCT TCA AAT CCG 865 Thr Thr Asp Leu Leu His Ser Glu Leu Val Asn Gly Ser Ser Asn Pro 55 60 65 ATG TTG GAT GCA CGT CCA GTT CGT AAG TCA GGG TAT ATT GTC ATT ACT 913 Met Leu Asp Ala Arg Pro Val Arg Lys Ser Gly Tyr Ile Val Ile Thr 70 75 80 85 TCA GAT AAG GGA TTA GTT GGA GGA TAT AAT TCA ACC ATT CTT AAA GCT 961 Ser Asp Lys Gly Leu Val Gly Gly Tyr Asn Ser Thr Ile Leu Lys Ala 90 95 100 GTC TTG GAT ATG ATT AAA CGT GAC CAT GAT TCT GAA GAT GAA TAT GCT 1009 Val Leu Asp Met Ile Lys Arg Asp His Asp Ser Glu Asp Glu Tyr Ala 105 110 115 ATC ATC TCT ATT GGT GGA ACA GGT TCA GAT TTC TTC AAA GCT CGT AAC 1057 Ile Ile Ser Ile Gly Gly Thr Gly Ser Asp Phe Phe Lys Ala Arg Asn 120 125 130 ATG AAT GTT GCT TTT GAA CTT CGT GGC CTT GAA GAT CAA CCT AGT TTC 1105 Met Asn Val Ala Phe Glu Leu Arg Gly Leu Glu Asp Gln Pro Ser Phe 135 140 145 GAT CAA GTC GGG GAA ATC ATT CTA AAA GCT GTA GGA ATG TAT CAA AAT 1153 Asp Gln Val Gly Glu Ile Ile Leu Lys Ala Val Gly Met Tyr Gln Asn 150 155 160 165 GAG CTT TTT GAT GAA CTT TAT GTG TGT TAC AAT CAT CAT ATT AAT AGT 1201 Glu Leu Phe Asp Glu Leu Tyr Val Cys Tyr Asn His His Ile Asn Ser 170 175 180 TTG TTT TGT GAA GTT TGT GTT GAA AAA ATG CTT CCA ATT GCT GAT TTT 1249 Leu Phe Cys Glu Val Cys Val Glu Lys Met Leu Pro Ile Ala Asp Phe 185 190 195 GAT CCT AAT GAA TTT GAA GGC CAT GTA TTG ACC AAG TTT GAA TTG GAA 1297 Asp Pro Asn Glu Phe Glu Gly His Val Leu Thr Lys Phe Glu Leu Glu 200 205 210 CCA AGT TGT GAT ACT ATT TTG GAT CAA CTT TTG CCC ACA ATA GTC GGT 1345 Pro Ser Cys Asp Thr Ile Leu Asp Gln Leu Leu Pro Thr Ile Val Gly 215 220 225 GAG AGT TTT ATC TAC GGT GCT ATC GTA GAT GCC AAA ACA GCT GAG CAT 1393 Glu Ser Phe Ile Tyr Gly Ala Ile Val Asp Ala Lys Thr Ala Glu His 230 235 240 245 GCT GCT GGT ATG ACC GCA ATG CAG ACT GCC ACT GAT AAT GCT AAG AAA 1441 Ala Ala Gly Met Thr Ala Met Gln Thr Ala Thr Asp Asn Ala Lys Lys 250 255 260 ATA ATT AAC GAT TTA ACA ATT CAA TAC AAC CGT GCA CGT CAA GCA GCC 1489 Ile Ile Asn Asp Leu Thr Ile Gln Tyr Asn Arg Ala Arg Gln Ala Ala 265 270 275 ATT ACT CAG GAA ATC ACT GAG ATT GTT GGC GGT GCT AGT GCA CTT GAA 1537 Ile Thr Gln Glu Ile Thr Glu Ile Val Gly Gly Ala Ser Ala Leu Glu 280 285 290 TAGCTAGAGA TTTGTCTTGA TTTGACATAC AATAAAAAGG GATGATTGTC ATCCAGAAAA 1597 CTTCATAAGG AGAAAACA ATG AGC TCA GGC AAA ATT GCT CAG GTT GTT GGT 1648 Met Ser Ser Gly Lys Ile Ala Gln Val Val Gly 1 5 10 CCT GTT GTA GAC GTA GCG TTT GCA ACT GGC GAT AAA CTT CCT GAG ATT 1696 Pro Val Val Asp Val Ala Phe Ala Thr Gly Asp Lys Leu Pro Glu Ile 15 20 25 AAC AAT GCA TTG GTC GTT TAC ACT GAG AAG AAA AGT CTT AGA CGG ATG 1744 Asn Asn Ala Leu Val Val Tyr Thr Glu Lys Lys Ser Leu Arg Arg Met 30 35 40 GTG CTC GAA GTA GCT TCG TTG AAA CTT GGA GAA GGT GTG GTT CGT ACC 1792 Val Leu Glu Val Ala Ser Leu Lys Leu Gly Glu Gly Val Val Arg Thr 45 50 55 ATT GCC ATG GAA TCT ACT GAT GGA TTG ACT CGT GGG CTA GAA GTT CTG 1840 Ile Ala Met Glu Ser Thr Asp Gly Leu Thr Arg Gly Leu Glu Val Leu 60 65 70 75 GAC ACA GGT CGT CCA ATC AGT GTT CCT GTT GGT AAA GAA CTT CTT GGA 1888 Asp Thr Gly Arg Pro Ile Ser Val Pro Val Gly Lys Glu Leu Leu Gly 80 85 90 CGT GTC TTT AAC GTG CTT GGA GAT ACC ATT GAC ATG GAA GCA CCT TTT 1936 Arg Val Phe Asn Val Leu Gly Asp Thr Ile Asp Met Glu Ala Pro Phe 95 100 105 GCA GAT GAT GCA GAG CGT GAA CCA ATT CAT AAA AAA GCA CCT ACC TTC 1984 Ala Asp Asp Ala Glu Arg Glu Pro Ile His Lys Lys Ala Pro Thr Phe 110 115 120 GAT GAA TTG TCA ACA AGT ACT GAA ATC CTT GAA ACA GGG ATT AAA GTT 2032 Asp Glu Leu Ser Thr Ser Thr Glu Ile Leu Glu Thr Gly Ile Lys Val 125 130 135 ATC GAC TTG CTT GCC CCT TAT CTT AAA GGT GGT AAA GTC GGA CTT TTC 2080 Ile Asp Leu Leu Ala Pro Tyr Leu Lys Gly Gly Lys Val Gly Leu Phe 140 145 150 155 GGT GGT GCC GGT GTT GGT AAG GCC GTT CTT ATT CAA GAG CTG AAT CAC 2128 Gly Gly Ala Gly Val Gly Lys Ala Val Leu Ile Gln Glu Leu Asn His 160 165 170 AAC ATT GCT CAA GAA CAC GGT GGC ATT TCC GTG 2161 Asn Ile Ala Gln Glu His Gly Gly Ile Ser Val 175 180 212 amino acids amino acid linear protein 11 Asp Ser His Leu His Ser Arg Leu Leu Glu Arg Ser Ala Lys Leu Ser 1 5 10 15 Asp Asp Leu Gly Gly Gly Ser Met Thr Ala Leu Pro Ile Ile Gln Thr 20 25 30 Gln Ala Gly Asp Ile Ser Ala Tyr Ile Ala Thr Asn Val Ile Ser Ile 35 40 45 Thr Asp Gly Gln Ile Phe Leu Gln Glu Asn Leu Phe Asn Ser Gly Ile 50 55 60 Arg Pro Ala Ile Asp Ala Gly Ser Ser Val Ser Arg Val Gly Gly Ser 65 70 75 80 Ala Gln Ile Lys Ala Met Lys Lys Val Ala Gly Thr Leu Arg Leu Asp 85 90 95 Leu Ala Ser His Arg Glu Leu Glu Ala Phe Thr Gln Phe Gly Ser Asp 100 105 110 Leu Asp Ala Ala Thr Gln Ala Lys Leu Asn Arg Gly Arg Arg Thr Val 115 120 125 Glu Val Leu Lys Gln Pro Leu His Asn Pro Leu Pro Val Glu Lys Gln 130 135 140 Val Leu Ile Leu Tyr Ala Leu Thr His Gly Phe Leu Asp Ser Val Pro 145 150 155 160 Val Asp Gln Ile Leu Asp Phe Glu Glu Ala Leu Tyr Asp Tyr Phe Asp 165 170 175 Ser His His Glu Asp Ile Phe Glu Thr Ile Arg Ser Thr Lys Asp Leu 180 185 190 Pro Glu Glu Ala Val Leu Asn Glu Ala Ile Gln Ala Phe Lys Asp Gln 195 200 205 Ser Glu Tyr Lys 210 293 amino acids amino acid linear protein 12 Met Ala Gly Ser Leu Arg Glu Ile Lys Ala Lys Ile Ala Ser Ile Lys 1 5 10 15 Gln Thr Ser His Ile Thr Gly Ala Met Gln Met Val Ser Ala Ser Lys 20 25 30 Leu Thr Arg Ser Glu Gln Ala Ala Lys Asp Phe Gln Ile Tyr Ala Ser 35 40 45 Lys Ile Arg Gln Ile Thr Thr Asp Leu Leu His Ser Glu Leu Val Asn 50 55 60 Gly Ser Ser Asn Pro Met Leu Asp Ala Arg Pro Val Arg Lys Ser Gly 65 70 75 80 Tyr Ile Val Ile Thr Ser Asp Lys Gly Leu Val Gly Gly Tyr Asn Ser 85 90 95 Thr Ile Leu Lys Ala Val Leu Asp Met Ile Lys Arg Asp His Asp Ser 100 105 110 Glu Asp Glu Tyr Ala Ile Ile Ser Ile Gly Gly Thr Gly Ser Asp Phe 115 120 125 Phe Lys Ala Arg Asn Met Asn Val Ala Phe Glu Leu Arg Gly Leu Glu 130 135 140 Asp Gln Pro Ser Phe Asp Gln Val Gly Glu Ile Ile Leu Lys Ala Val 145 150 155 160 Gly Met Tyr Gln Asn Glu Leu Phe Asp Glu Leu Tyr Val Cys Tyr Asn 165 170 175 His His Ile Asn Ser Leu Phe Cys Glu Val Cys Val Glu Lys Met Leu 180 185 190 Pro Ile Ala Asp Phe Asp Pro Asn Glu Phe Glu Gly His Val Leu Thr 195 200 205 Lys Phe Glu Leu Glu Pro Ser Cys Asp Thr Ile Leu Asp Gln Leu Leu 210 215 220 Pro Thr Ile Val Gly Glu Ser Phe Ile Tyr Gly Ala Ile Val Asp Ala 225 230 235 240 Lys Thr Ala Glu His Ala Ala Gly Met Thr Ala Met Gln Thr Ala Thr 245 250 255 Asp Asn Ala Lys Lys Ile Ile Asn Asp Leu Thr Ile Gln Tyr Asn Arg 260 265 270 Ala Arg Gln Ala Ala Ile Thr Gln Glu Ile Thr Glu Ile Val Gly Gly 275 280 285 Ala Ser Ala Leu Glu 290 182 amino acids amino acid linear protein 13 Met Ser Ser Gly Lys Ile Ala Gln Val Val Gly Pro Val Val Asp Val 1 5 10 15 Ala Phe Ala Thr Gly Asp Lys Leu Pro Glu Ile Asn Asn Ala Leu Val 20 25 30 Val Tyr Thr Glu Lys Lys Ser Leu Arg Arg Met Val Leu Glu Val Ala 35 40 45 Ser Leu Lys Leu Gly Glu Gly Val Val Arg Thr Ile Ala Met Glu Ser 50 55 60 Thr Asp Gly Leu Thr Arg Gly Leu Glu Val Leu Asp Thr Gly Arg Pro 65 70 75 80 Ile Ser Val Pro Val Gly Lys Glu Leu Leu Gly Arg Val Phe Asn Val 85 90 95 Leu Gly Asp Thr Ile Asp Met Glu Ala Pro Phe Ala Asp Asp Ala Glu 100 105 110 Arg Glu Pro Ile His Lys Lys Ala Pro Thr Phe Asp Glu Leu Ser Thr 115 120 125 Ser Thr Glu Ile Leu Glu Thr Gly Ile Lys Val Ile Asp Leu Leu Ala 130 135 140 Pro Tyr Leu Lys Gly Gly Lys Val Gly Leu Phe Gly Gly Ala Gly Val 145 150 155 160 Gly Lys Ala Val Leu Ile Gln Glu Leu Asn His Asn Ile Ala Gln Glu 165 170 175 His Gly Gly Ile Ser Val 180 914 base pairs nucleic acid double linear DNA (genomic) NO NO C-terminal Phaffia rhodozyma CDS 51..824 /partial /codon_start= 51 /product= “ATPase subunit, partial sequence” /gene= “ATP2” /standard_name= “beta subunit of the F1 portion of the F0F1 ATPase” /label= beta-subunit 14 GAATTCTCAA CCTTGAGGGT GACTCCAAGG TCGCTCTTGT CTTCGGACAG ATG AAC 56 Met Asn GAG CCC CCG GGT GCT CGA GCC CGA GTC GCT TTG ACT GGT TTG ACC ATC 104 Glu Pro Pro Gly Ala Arg Ala Arg Val Ala Leu Thr Gly Leu Thr Ile 185 190 195 200 GCC GAG TAC TTC CGA GAC GAG GAA GGA CAG GAT GTC TTG CTT TTC ATC 152 Ala Glu Tyr Phe Arg Asp Glu Glu Gly Gln Asp Val Leu Leu Phe Ile 205 210 215 GAC AAC ATT TTC CGA TTC ACC CAG GCC GGT TCT GAG GTG TCT GCC TTG 200 Asp Asn Ile Phe Arg Phe Thr Gln Ala Gly Ser Glu Val Ser Ala Leu 220 225 230 CTT GGT CGA ATT CCC TCC GCC GTC GGA TAC CAG CCC ACT CTT TCC ACC 248 Leu Gly Arg Ile Pro Ser Ala Val Gly Tyr Gln Pro Thr Leu Ser Thr 235 240 245 GAT ATG GGA GGT ATG CAG GAG CGA ATT ACC ACC ACC AAG AAG GGA TCC 296 Asp Met Gly Gly Met Gln Glu Arg Ile Thr Thr Thr Lys Lys Gly Ser 250 255 260 ATC ACT TCC GTC CAG GCC GTC TAC GTG CCT GCT GAT GAT TTG ACC GAT 344 Ile Thr Ser Val Gln Ala Val Tyr Val Pro Ala Asp Asp Leu Thr Asp 265 270 275 280 CCT GCC CCC GCC ACC ACC TTC GCC CAC TTG GAC GCC ACC ACT GTG TTG 392 Pro Ala Pro Ala Thr Thr Phe Ala His Leu Asp Ala Thr Thr Val Leu 285 290 295 TCT CGA GGT ATC GCT GAG TTG GGT ATC TAC CCC GCT GTC GAT CCC CTT 440 Ser Arg Gly Ile Ala Glu Leu Gly Ile Tyr Pro Ala Val Asp Pro Leu 300 305 310 GAT TCT AAG TCC CGA ATG CTC GAC CCC CGA ATT GTC GGA CAG GAG CAC 488 Asp Ser Lys Ser Arg Met Leu Asp Pro Arg Ile Val Gly Gln Glu His 315 320 325 TAC GAC ATC GCC ACC AAG ACC CAG AAG ATC CTC CAG GAC TAC AAG TCC 536 Tyr Asp Ile Ala Thr Lys Thr Gln Lys Ile Leu Gln Asp Tyr Lys Ser 330 335 340 CTC CAG GAT ATC ATT GCC ATT CTT GGT ATG GAT GAG TTG TCT GAG GAG 584 Leu Gln Asp Ile Ile Ala Ile Leu Gly Met Asp Glu Leu Ser Glu Glu 345 350 355 360 GAC AAG TTG ACC GTC GAG CGA GCC CGA AAG ATC CAG CGA TTC ATG TCG 632 Asp Lys Leu Thr Val Glu Arg Ala Arg Lys Ile Gln Arg Phe Met Ser 365 370 375 CAG CCT TTC GCT GTC GCT CAG GTC TTC ACT GGT ATC GAG GGA AAG CTT 680 Gln Pro Phe Ala Val Ala Gln Val Phe Thr Gly Ile Glu Gly Lys Leu 380 385 390 GTT CCC TTG AAG ACT ACT TTG GAG TCC TTT AAG GAG CTT CTT TCC GGA 728 Val Pro Leu Lys Thr Thr Leu Glu Ser Phe Lys Glu Leu Leu Ser Gly 395 400 405 GCC TGC GAC CAC CTC CCT GAG TCT GCT TTC TAC ATG GTT GGT GAC ATC 776 Ala Cys Asp His Leu Pro Glu Ser Ala Phe Tyr Met Val Gly Asp Ile 410 415 420 GCT GAT GTC AAG GCC AAG GCT GCT GCC CAG GCT AAG GAG TTG GCT GCT 824 Ala Asp Val Lys Ala Lys Ala Ala Ala Gln Ala Lys Glu Leu Ala Ala 425 430 435 440 TAAGAGAAGA GTTGTCGAAT GTGTTTCGAG GTGTCAGAGT TGTCTTTTAT GAATGTTTCT 884 ATCTCCTTAA AAAAAAAAAA AAAAAAAAAA 914 258 amino acids amino acid linear protein 15 Met Asn Glu Pro Pro Gly Ala Arg Ala Arg Val Ala Leu Thr Gly Leu 1 5 10 15 Thr Ile Ala Glu Tyr Phe Arg Asp Glu Glu Gly Gln Asp Val Leu Leu 20 25 30 Phe Ile Asp Asn Ile Phe Arg Phe Thr Gln Ala Gly Ser Glu Val Ser 35 40 45 Ala Leu Leu Gly Arg Ile Pro Ser Ala Val Gly Tyr Gln Pro Thr Leu 50 55 60 Ser Thr Asp Met Gly Gly Met Gln Glu Arg Ile Thr Thr Thr Lys Lys 65 70 75 80 Gly Ser Ile Thr Ser Val Gln Ala Val Tyr Val Pro Ala Asp Asp Leu 85 90 95 Thr Asp Pro Ala Pro Ala Thr Thr Phe Ala His Leu Asp Ala Thr Thr 100 105 110 Val Leu Ser Arg Gly Ile Ala Glu Leu Gly Ile Tyr Pro Ala Val Asp 115 120 125 Pro Leu Asp Ser Lys Ser Arg Met Leu Asp Pro Arg Ile Val Gly Gln 130 135 140 Glu His Tyr Asp Ile Ala Thr Lys Thr Gln Lys Ile Leu Gln Asp Tyr 145 150 155 160 Lys Ser Leu Gln Asp Ile Ile Ala Ile Leu Gly Met Asp Glu Leu Ser 165 170 175 Glu Glu Asp Lys Leu Thr Val Glu Arg Ala Arg Lys Ile Gln Arg Phe 180 185 190 Met Ser Gln Pro Phe Ala Val Ala Gln Val Phe Thr Gly Ile Glu Gly 195 200 205 Lys Leu Val Pro Leu Lys Thr Thr Leu Glu Ser Phe Lys Glu Leu Leu 210 215 220 Ser Gly Ala Cys Asp His Leu Pro Glu Ser Ala Phe Tyr Met Val Gly 225 230 235 240 Asp Ile Ala Asp Val Lys Ala Lys Ala Ala Ala Gln Ala Lys Glu Leu 245 250 255 Ala Ala 375 base pairs nucleic acid double linear DNA (genomic) Trichoderma reesei CDS 50..361 /partial /codon_start= 50 /product= “ATPase subunit, partial sequence” /gene= “ATP2” /standard_name= “beta subunit of F1 portion of the F0F1 ATPase” /label= beta-subunit 16 TACTCGAAGA ATTCGGCACG AGGCTGATTG CTCTCGGTCA TCTGCCAAG ATG TTC 55 Met Phe 260 AAG AGC GGC GTT TCG TCC CTC GCC AGG GCT GCC CGC CCA TCA ATT ACC 103 Lys Ser Gly Val Ser Ser Leu Ala Arg Ala Ala Arg Pro Ser Ile Thr 265 270 275 GCT CGA CGA GCT ATC CGA CCA GCC TTC CCT CGA ACC CCC CTC GCG AGG 151 Ala Arg Arg Ala Ile Arg Pro Ala Phe Pro Arg Thr Pro Leu Ala Arg 280 285 290 CTT GCC AGC ACC CAG AGC GTC GGA GAT GGC AAG ATC CAC CAG GTC ATT 199 Leu Ala Ser Thr Gln Ser Val Gly Asp Gly Lys Ile His Gln Val Ile 295 300 305 GGT GCC GTC GTC GAC GTC AAG TTC GAC ACC GCC AAG CTG CCT CCT ATC 247 Gly Ala Val Val Asp Val Lys Phe Asp Thr Ala Lys Leu Pro Pro Ile 310 315 320 CTG AAC GCC CTG GAG ACC ACC AAC AAC AAC CAG AAG CTG GTC CTC GAG 295 Leu Asn Ala Leu Glu Thr Thr Asn Asn Asn Gln Lys Leu Val Leu Glu 325 330 335 340 GTG GCT CAA CAC TTG GGC GAG AAT GTC GTT CGC TGC ATT GCC ATG GAC 343 Val Ala Gln His Leu Gly Glu Asn Val Val Arg Cys Ile Ala Met Asp 345 350 355 GGA TCC GAG GGT CTC GTC GTGGTTCCAA GGCA 375 Gly Ser Glu Gly Leu Val 360 104 amino acids amino acid linear protein 17 Met Phe Lys Ser Gly Val Ser Ser Leu Ala Arg Ala Ala Arg Pro Ser 1 5 10 15 Ile Thr Ala Arg Arg Ala Ile Arg Pro Ala Phe Pro Arg Thr Pro Leu 20 25 30 Ala Arg Leu Ala Ser Thr Gln Ser Val Gly Asp Gly Lys Ile His Gln 35 40 45 Val Ile Gly Ala Val Val Asp Val Lys Phe Asp Thr Ala Lys Leu Pro 50 55 60 Pro Ile Leu Asn Ala Leu Glu Thr Thr Asn Asn Asn Gln Lys Leu Val 65 70 75 80 Leu Glu Val Ala Gln His Leu Gly Glu Asn Val Val Arg Cys Ile Ala 85 90 95 Met Asp Gly Ser Glu Gly Leu Val 100 

What is claimed is:
 1. A method of improving the production of biomass or a desired product from a cell, characterized by expressing an uncoupled ATPase activity in said cell to induce conversion of ATP to ADP without primary effects on other cellular metabolites or functions, and incubating the cell with a suitable substrate to produce said biomass or product.
 2. A method according to claim 1, characterized by expressing in said cell the soluble part (F₁) of the membrane bound (F₀F₁ type) H⁺-ATPase or a portion of F₁ exhibiting ATPase activity.
 3. A method according to claim 1, wherein said cell is a prokaryotic cell.
 4. A method according to claim 3, wherein said cell is selected from the group consisting of bacteria belonging to the genera Lactococcus, Streptococcus, Enterococcus, Lactobacillus, Leuconostoc, Escherichia, Zymomonas, Bacillus and Pseudamonas.
 5. A method according to claim 1, wherein said cell is a eukaryotic cell.
 6. A method according to claim 5, wherein said cell is a yeast cell.
 7. A method according to claim 6, wherein said cell belongs to Saccharomyces cerevisiae or TriChoderma reesei.
 8. A method according to claim 1, wherein said cell is transformed or transfected with an expression vector including DNA encoding F₁ or a portion thereof exhibiting ATPase activity under the control of a promoter functioning in said cell, and said DNA is expresses in the cell.
 9. A method according to claim 8, wherein said DNA encoding F₁ or a portion thereof is homologous to said cell.
 10. A method according to claim 8, wherein said DNA encoding F₁ or a portion thereof is heterologous to said cell.
 11. A method according to claim 8, wherein said DNA encoding F₁ or a portion thereof is derived from a prokaryotic organism.
 12. A method according to claim 11, wherein said DNA encoding F₁or a portion thereof is derived from Escherichia coli, Lactococcus lactis or Streptococcus thermophilus and is selected from the group consisting of the gene encoding the F₁ subunit β or a portion thereof and various combinations of said gene or portion with the genes encoding the F₁ subunits δ, α, γ and ε or portions thereof.
 13. A method according to claim 12, wherein said DNA encoding F₁ or a portion thereof is selected from the group consisting of the Escherichia coli, Streptococcus thermophilus and Lactococcus lactis genes atpHAGDC (coding for subunits δ, α, γ, β, ε), atpAGDC (coding for subunits α, γ, β, ε), atpAGD (coding for subunits α, γ, β, ε), atpDC (coding for subunits β, ε) and atpD (coding for subunit β alone).
 14. A method according to claim 8, wherein said DNA encoding F₁ or a portion thereof is derived from a eukaryotic organism.
 15. A method according to claim 14, wherein said DNA encoding F₁ or a portion thereof is derived from Saccharomyces cerevisiae, Phaffia rhodozyma or Trichoderma reesei and is selected from the group consisting of the gene encoding the F₁ subunit β or a portion thereof and various combinations of said gene or portion with the genes encoding the other F₁ subunits or portions thereof.
 16. A vector including DNA encoding the soluble part (F₁) of the membrane bound (F₀F₁ type) H⁺-ATPase or a portion of F₁ exhibiting ATPase activity, said DNA being derived from Lactococcus lactis subsp. cremoris and having the sequence stated in SEQ ID No.
 1. 17. A vector including DNA encoding the soluble part (F₁) of the membrane bound (F₀F₁ type) H⁺-ATPase or a portion of F₁ exhibiting ATPase activity, said DNA being derived from Lactococcus lactis subsp. lactis and having the sequence stated in SEQ ID No.
 6. 18. A vector including DNA encoding the soluble part (F₁) of the membrane bound (F₀F₁ type) H⁺-ATPase or a portion of F₁ exhibiting ATPase activity, said DNA being derived from Streptococcus thermophilus and having the sequence stated in SEQ ID No.
 10. 19. A vector including DNA encoding the soluble part (F₁) of the membrane bound (F₀F₁ type) H⁺-ATPase or a portion of F₁ exhibiting ATPase activity, said DNA being derived from Phaffia rhodozyma and having the sequence stated in SEQ ID No.
 14. 20. A vector including DNA encoding the soluble part (F₁) of the membrane bound (F₀F₁ type) H⁺-ATPase or a portion of F₁ exhibiting ATPase activity, said DNA being derived from Trichoderma reesei and having the sequence stated in SEQ ID No.
 16. 21. An expression vector including DNA as defined in any one of claims 16-20 under the control of a promoter capable of directing the expression of said DNA in a prokaryotic or eukaryotic cell.
 22. A method of optimizing the formation of biomass or a desired product by a cell, characterized by expressing different levels of uncoupled ATPase activity in the cell, incubating the cell on a suitable substrate, measuring the conversion rate of substrate into biomass or the desired product at each level of ATPase expression, and choosing a level of ATPase expression at which the conversion rate is optimized.
 23. A method according to claim 22, wherein a number of specimens of said cell are transformed or transfected with their respective expression vector each including DNA encoding a different portion of the cytoplasmic part (F₁) of the membrane bound (F₀F₁ type) H⁺-ATPase up to and including the entire F₁, each portion exhibiting ATPase activity, said DNA in each expression vector being under the control of a promoter functioning in said cell, incubating each cell specimen on a suitable substrate, measuring the conversion rate of substrate into biomass or the desired product by each specimen, and choosing a specimen yielding an optimized conversion rate.
 24. A method according to claim 22, wherein a number of specimens of said cell are transformed or transfected with their respective expression vector including DNA encoding a portion of the cytoplasmic part (F₁) of the membrane bound (F₀F₁ type) H⁺-ATPase up to and including the entire F₁, said portion exhibiting ATPase activity, said DNA in the respective expression vectors being under the control of each of a series of promoters covering a broad range of promoter activities and functioning in said cell, incubating each cell specimen on a suitable substrate, measuring the conversion rate of substrate into biomass or the desired product by each specimen, and choosing a specimen yielding an optimized conversion rate.
 25. A method according to claim 24, wherein the respective expression vectors include DNA encoding different such portions of F₁ up to and including the entire F₁, each DNA in respective expression vectors being under the control of each of a series of promoters covering a broad range of promoter activities and functioning in said cell.
 26. A method according to any one of claims 23-25, wherein the promoter in each expression vector is an inducible promoter, and each cell specimen is grown at different concentrations of inducer.
 27. A method according to any one of claims 23-25, wherein said DNA encoding a portion of F₁ up to and including the entire F₁ is homologous to said cell.
 28. A method according to any one of claims 23-25, wherein said DNA encoding a portion of F₁ up to and including the entire F₁ is heterologous to said cell.
 29. A method according to any one of claims 23-25 wherein said DNA encoding a portion of F₁ up to and including the entire F₁ is derived from a prokaryotic organism.
 30. A method according to claim 29, wherein said DNA encoding a portion of F₁ up to and including the entire F₁ is derived from Escherichia coli, Lactococcus lactis or Streptococcus thermophilus and is selected from the group consisting of the gene encoding the F₁ subunit β or a portion thereof and various combinations of said gene or to portion with the genes encoding the F₁ subunits δ, α, γ and ε r portions thereof.
 31. A method according to claim 30, wherein said DNA encoding a portion of F₁ up to and including the entire F₁ is selected from the group consisting of the E. coli genes atpAGDC (coding for subunits α, γ, β, ε), atpAGD (coding for subunits α, γ, β, atpDC (coding for subunits β, ε) and atpD (coding for subunit β alone).
 32. A method according to any one of claims 23-25, wherein said DNA encoding a portion of F₁ up to and including the entire F₁ is derived from a eukaryotic organism.
 33. A method according to claim 32, wherein said DNA encoding F₁ or a portion thereof is derived from Saccharomyces cerevisiae, Phaffia rhodozyma or Trichoderma reesei and is selected from the group consisting of the gene encoding the F₁ subunit β or a portion thereof and various combinations of said gene or portion with the genes encoding the other F₁ subunits or portions thereof. 